Aspartase mutant and application thereof in L-aspartic acid conversion

Abstract

The invention discloses an aspartase mutant and an application of the aspartase mutant in conversion of L-aspartic acid. Compared with an amino acid sequence as shown in SEQ ID NO: 1, the aspartase mutant disclosed by the invention contains one or more mutations in G410C, M90H or K126R. Compared with an original enzyme, the obtained aspartase mutant has higher enzyme activity, and the enzyme activity of the aspartase mutant is more stable in a constant-temperature accelerated storage experiment. The time for converting the fumaric acid substrate by the aspartase mutant in a system above tonnages is short, and a foundation is laid for industrial production of L-aspartic acid.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to an aspartase mutant and its application. Background Art

[0002] L-aspartic acid (L-Asp), also known as L-asparagine, is one of the 20 amino acids that make up proteins. It is widely used in industries such as chemical engineering, food, and medicine. It is an important industrial raw material with great potential for market development. The main production methods include traditional fermentation method, bioenzyme method, etc. The traditional fermentation method uses glucose as a carbon source and uses microorganisms to ferment and produce L-aspartic acid. The fermentation method was the main method for the early industrial production of L-aspartic acid. Its production cycle is long, there are many by-products, the production cost is relatively high, and the technical risk is large, which limits the application of L-aspartic acid products in the pharmaceutical field at that time. The production of L-aspartic acid by the bioenzyme method uses fumaric acid and ammonia as raw materials and uses L-aspartase produced by fermentation to directly convert the raw materials to produce L-aspartic acid. Therefore, the process technology using fumaric acid as a raw material is the mainstream technology of the current L-aspartic acid production process.

[0003] Aspartase belongs to the ammonia lyase family. It was first discovered in several facultative aerobic bacteria and was later confirmed to be widely present in various bacteria, plants, mammals, and viruses. Currently, the sources of L-aspartase mainly include Proteus vulgaris (P. vulgaris) and Escherichia coli (Escherichia coli). Among them, the aspartase aspA from Escherichia coli has been confirmed to have good catalytic activity. AspA is composed of 4 subunits of 50 kd, each subunit encodes 478 amino acid residues, and the molecular weight is about 200 kd, showing a typical P21212 symmetry. A single subunit has no catalytic function. Each subunit is composed of 3 domains (D1, D2, and D3) in an S shape. Among them, the central domain D2 is the amino acids at positions 142-396. It is the most conserved domain of aspartase. The active site of the wild-type aspartase located in the D2 domain has been determined in early studies to be T187, M321, K324, K326. And aspartase has a high specificity for the substrate fumaric acid. However, since aspartase catalyzes a reversible reaction, there is still a problem of insufficient conversion rate of fumaric acid in the whole process. Therefore, it is urgent to develop a highly active aspartase by genetic engineering means to improve the conversion rate of the substrate and apply it to the industrial production of L-aspartic acid.

[0004] CN108546698B discloses an aspartase mutant. When used in the large-scale production of L-aspartic acid, in a reaction system of 50 L and a fumaric acid substrate concentration of 400 g / L, with an enzyme addition amount of 4000 (U / g substrate), the conversion rate can reach 98.5% in 24 h. However, when this aspartase mutant is used in large-scale production, the conversion efficiency is low and the conversion time is long.

[0005] CN102559538A (publication date: July 11, 2012) obtained an Escherichia coli strain with high-yield aspartase through mutagenesis. Its enzyme activity in shake flask fermentation is 4200 U / ml, and in 2 m 3 the enzyme activity in seed tank fermentation is 4500 U / ml. When the fumaric acid substrate concentration is 20.0%, in 20 m 3 the bioconversion time in the tank is 6 hours, and the conversion rate reaches 97.5%.

[0006] US6015704A (publication date: January 18, 2000) discloses aspartase mutants and their applications. Specifically, the histidines at positions 25, 123, 421, and 463 of aspartase are mutated to glutamine, and the amino acids at positions 471 - 477 are deleted, respectively obtaining Gln 25 、Gln 123 、Gln 421 、Gln 463 、Ochre 471 、Gln 25 & Ochre 471 mutants. The highest enzyme activity of the Gln 25 & Ochre 471 mutant is 90.77 U / mg, which is 3.23 times higher than that of the starting strain. However, when applied to the actual production of aspartic acid, the enzyme activity needs to be improved. SUMMARY OF THE INVENTION

[0007] To solve the defects of low aspartase activity, long conversion time, and easy inactivation of the enzyme during long-term storage in the existing technology for aspartic acid production, the present invention provides an aspartase mutant and its application in L-aspartic acid conversion through rational design and screening of the enzyme.

[0008] One aspect of the present invention provides an aspartase mutant, which contains one or more mutations of G410C, K126R, or M90H compared with the amino acid sequence shown in SEQ ID NO: 1.

[0009] G410C means that the amino acid at position 410 is mutated from G to C, K126R means that the amino acid at position 126 is mutated from K to R, and M90H means that the amino acid at position 90 is mutated from M to H.

[0010] In some embodiments, the aspartase mutant comprises M90H, K126R, G410C, M90H / K126R / G410C, M90H / G410C, K126R / G410C or M90H / K126R.

[0011] M90H / K126R represents a double mutant comprising M90H and K126R, and M90H / K126R / G410C represents a triple mutant comprising M90H, K126R and G410C.

[0012] In some specific embodiments, the aspartase mutant comprises M90H / K126R / G410C, M90H / G410C, K126R / G410C or M90H / K126R.

[0013] In some more specific embodiments, the aspartase mutant comprises M90H / K126R / G410C.

[0014] On the other hand, the present invention provides an isolated nucleic acid encoding the above-mentioned aspartase mutant.

[0015] On the other hand, the present invention provides a recombinant expression vector comprising the above-mentioned isolated nucleic acid.

[0016] In some embodiments, the backbone of the recombinant expression vector is a plasmid such as pET-28a, pUC19, pBAD, pKD3, pTrc99a, pBR322, pDG148 or pPIC9.

[0017] On the other hand, the present invention provides a transformant comprising the above-mentioned isolated nucleic acid or the above-mentioned recombinant expression vector.

[0018] In some embodiments, the host cells used in constructing the transformant are Escherichia coli, Bacillus subtilis and Bacillus licheniformis.

[0019] In some specific embodiments, the Escherichia coli is Escherichia coli BL21(DE3).

[0020] On the other hand, the present invention provides a method for preparing the above-mentioned aspartase mutant, comprising culturing the above-mentioned transformant to obtain a fermentation product, and obtaining the aspartase mutant from the fermentation product.

[0021] On the other hand, the present invention provides a method for producing aspartic acid, which is produced by using the above-mentioned aspartase mutant or the above-mentioned transformant.

[0022] In some specific embodiments, fumaric acid is used as a substrate.

[0023] On the other hand, the present invention provides the use of the above-mentioned aspartase mutant, the above-mentioned isolated nucleic acid, the above-mentioned recombinant expression vector or the above-mentioned transformant in the production of aspartic acid.

[0024] Based on the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0025] The reagents and raw materials used in the present invention are all commercially available.

[0026] The positive and progressive effects of the present invention are as follows:

[0027] The enzyme activity of the aspartase mutant screened by the technical scheme of the present invention is significantly higher than that of the original enzyme. The aspartase mutant has at least a 3-fold increase compared to the original enzyme, and the aspartase mutant is more stable in the enzyme activity during the constant temperature accelerated storage experiment. Among them, the triple mutant has a 13-fold increase compared to the original enzyme. In a system with a tonnage or more, the conversion time of the fumaric acid substrate is short, and the conversion time is only 4-5 hours, and the conversion efficiency is as high as over 99%, laying a foundation for the industrial production of L-aspartic acid. Description of the Drawings

[0028] Figure 1 It is for the study of the storage stability of liquid enzyme at 4±1°C.

[0029] Figure 2 It is for the study of the storage stability of liquid enzyme at -20±1°C. Detailed Embodiments

[0030] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0031] In this article, "L-aspartic acid" can be abbreviated as "aspartic acid" or "asparagine".

[0032] The transformant host of the present invention can be any microorganism suitable for expressing the aspartase mutant, including bacteria and fungi. The microorganism can be Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, and Bacillus licheniformis. Escherichia coli can be Escherichia coli BL21(DE3).

[0033] When used as a biocatalyst for the production of aspartic acid, the aspartase of the present invention can be in the form of an enzyme or a cell. The form of the enzyme includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, enzyme immobilized on a carrier, etc.; the form of the cell includes viable cells and dead cells.

[0034] Other methods can be used to obtain aspartase mutants, such as screening by directed evolution through error-prone PCR or DNA shuffling.

[0035] Ochre in US6015704A 471 The amino acid sequence of the mutant is SEQ ID NO:1 of the present invention, Gln 25 & Ochre 471 The mutant is H26Q in the present invention.

[0036] Example 1 Rational design of the mutation site of aspartase aspA1

[0037] First, submit the amino acid sequence SEQ ID NO:1 of aspartase aspA1 from Escherichia coli to the SWISS-MODEL server, select the model with high homology, high score and sequence similarity greater than 90% as the template for homology modeling, and conduct model evaluation.

[0038] GMQE is a quality assessment based on the binding properties of the target-template alignment, with a value between 0 and 1. The closer the value is to 1, the closer the model is to the experimental results. QMEAN is the scoring value of the model quality estimate. QMEAN > -4 is acceptable, and > -2.5 is credible. The GMQE we obtained is 0.87 and the QMEAN is -0.62, with a credible value. Therefore, a highly accurate simulated structure of aspartase is obtained.

[0039] Using the AutoDockTools software, perform molecular docking calculations on the highly accurate simulated structure of aspartase and the substrate fumaric acid for producing L-aspartic acid. Among the 50 docking conformations obtained, 6 are consistent with the original crystal complex. Select one of the conformations and expand it with pymol to analyze the amino acid interaction forces between aspartase and the substrate fumaric acid within range, including hydrogen bonds (the bond length of the hydrogen bond is the bond angle is greater than 60°), electrostatic interactions and the active center. By analyzing the simulated structure, simulate mutations to mutate methionine at position 90, lysine at position 126, and glycine at position 410, which are closer to the active center, into histidine, arginine, and cysteine respectively, increasing the hydrogen bond interaction between the enzyme molecule and the substrate, improving the substrate affinity, and at the same time reducing the activation energy of the catalytic reaction, obtaining an aspartase mutant with significantly improved interaction with the substrate fumaric acid. The amino acid sequence of the obtained aspartase mutant is SEQ ID NO:2. On the basis of SEQ ID NO:1, mutate histidine at position 26 into glutamine to construct the same mutant as in Comparative Document US6015704A (Gln 25 & Ochre 471)Compare under the same conditions.

[0040] Construction of Recombinant Expression Vector pET-aspA1 in Example 2

[0041] The aspA1 gene from Escherichia coli K-12 was selected, with the gene sequence SEQ ID NO:3, sent to Wuhan Kingcare Bioengineering Co., Ltd. for synthesis, and directly ligated to the pET-28a plasmid through the restriction enzyme sites BamHI and EcoRI to construct the recombinant expression vector pET-aspAO.

[0042] Construction of Recombinant Plasmids Containing Mutation Sites in Example 3

[0043] Based on the rationally designed sites in Example 1, the following mutant primers were designed and synthesized:

[0044] Table 1: Primer Table

[0045] Serial number Primer Sequence (5'-3') SEQ ID NO:4 Fp:M90H cggaaaatgccacgatcagttcccg SEQ ID NO:5 Rp:M90H cgggaactgatcgtggcattttccg SEQ ID NO:6 Fp:K126R ggtactgatattcaccacgttggtgacccatcag SEQ ID NO:7 Rp:K126R ctgatgggtcaccaacgtggtgaatatcagtacc SEQ ID NO:8 Fp:G410C caactctatctgcatcgttacttacctgaa SEQ ID NO:9 Rp:G410C ttcaggtaagtaacgatgcagatagagttg SEQ ID NO:10 Fp:H26Q tatggtgttcaaactctgagagcgat SEQ ID NO:11 Rp:H26Q tcagagtttgaacaccatagtaggca

[0046] Using the recombinant expression vector pET-aspAO as a template, the first-round reverse amplification was carried out with Fp:M90H / Rp:M90H, Fp:K126R / Rp:K126R, Fp:G410C / Rp:G410C, Fp:H26Q / Rp:H26Q as primers respectively. The amplification system was 2 μL of each upstream and downstream primer, 1 μL of DNA template, 25 μL of 2×Q5 Master Mix, and made up to 50.0 μL with ddH2O. The PCR program was: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 30 s, annealing at 55°C for 10 s, extension at 72°C for 3 min, 33 cycles, extension at 72°C for 10 min. The PCR products were verified by 1% agarose gel electrophoresis, and after DpnI digestion of the products, they were transformed into DH5α competent cells. Screening was carried out on kanamycin-resistant plates, and the plasmids were extracted and sent for sequencing to obtain four correct single-mutant recombinant plasmids pET-aspAM (M90H), pET-aspAK (K126R), pET-aspAG (G410C), and pET-aspAH (H26Q).

[0047] Using the single mutant recombinant plasmid pET-aspAM as a template, perform the second-round reverse amplification with Fp:K126R / Rp:K126R and Fp:G410C / Rp:G410C as primers respectively. At the same time, use pET-aspAK(K126R) as a template and Fp:G410C / Rp:G410C as primers for the second-round reverse amplification. The amplification system is 2 μL of each upstream and downstream primer, 1 μL of DNA template, 25 μL of 2×Q5 MasterMix, and make up to 50.0 μL with ddH2O. PCR program: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 30 s, annealing at 55°C for 10 s, extension at 72°C for 3 min, 33 cycles, extension at 72°C for 10 min. Verify the PCR products by 1% agarose gel electrophoresis, digest the products with DpnI, and then transform them into DH5α competent cells. Screen through kanamycin-resistant plates, extract plasmids and send them for sequencing to obtain the combined mutant recombinant plasmids pET-aspAMK(M90H / K126R), pET-aspAMG(M90H / G410C), and pET-aspAKG(K126R / G410C) with pairwise mutations.

[0048] Using the recombinant plasmid pET-aspAMK(M90H / K126R) as a template and Fp:G410C / Rp:G410C as primers for the third-round reverse amplification. The amplification system is 2 μL of each upstream and downstream primer, 1 μL of DNA template, 25 μL of 2×Q5 Master Mix, and make up to 50.0 μL with ddH2O. PCR program: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 30 s, annealing at 55°C for 10 s, extension at 72°C for 3 min, 33 cycles, extension at 72°C for 10 min. Verify the PCR products by 1% agarose gel electrophoresis, digest the products with DpnI, and then transform them into DH5α competent cells. Screen through kanamycin-resistant plates, extract plasmids and send them for sequencing to obtain the correct triple mutant recombinant plasmid pET-aspAMKG(M90H / K126R / G410C).

[0049] Example 4 Engineering Bacteria Expressing Aspartase and Aspartase Mutants

[0050] Using Escherichia coli BL21(DE3) as the host cell, introduce the above recombinant plasmids containing the original aspartase gene and its mutant genes into Escherichia coli BL21(DE3) competent cells by chemical transformation or electroporation, and obtain Escherichia coli recombinant bacteria containing aspartase or its mutants through resistance screening.

[0051] The Escherichia coli expressing the aspartase and its mutants constructed in Example 4 was cultured using a conventional E. coli LB medium and an aspartase fermentation medium. The formula of the LB medium is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. The formula of the aspartase fermentation medium is as follows: yeast peptone 10 g / L, yeast extract 5 g / L, glycerol 15 g / L, potassium dihydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 1.0 g / L, anhydrous sodium sulfate 1.8 g / L, ammonium sulfate 5.8 g / L, kanamycin sulfate 50 mg / L. The recombinant E. coli containing aspartase or its mutant was first inoculated into the LB medium and cultured at 37 °C and 180 rpm for 15 - 16 h. Then, the cultured fermentation broth was inoculated into the aspartase fermentation medium at an inoculation amount of 5% - 10% and fermented in a 15 L tank by fed-batch fermentation for about 7 - 9 h. When the cell concentration was about 50 g / L, the temperature was lowered to 24 - 26 °C, and IPTG was added at a final concentration of 0.1 mM for induction. After 24 h, when the cell concentration no longer increased, the fermentation was ended, and the cells were collected.

[0052] Determination of enzyme activity: The above fermentation broth was centrifuged at 12000 rpm for 20 min, and the supernatant was discarded to obtain the cells. 2.5 g of the cells were taken and resuspended in 25 mL of 0.1 M phosphate buffer at pH 7.0 - 7.2 to a cell concentration of 100 g / L. The conditions for ultrasonic disruption were as follows: disruption time 30 min, working for 3 s, interval 7 s, power 300 w. After disruption, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was taken and stored at -20 °C for later use. 1 mL of the above supernatant was added to 100 mL of 20% fumaric acid reaction solution (pH 8.5 - 8.6), and the reaction was carried out at 38 °C and 80 - 90 rpm for 10 min. 1 mL of the reaction solution was taken, boiled, and the aspartic acid concentration in the liquid phase was detected to calculate the enzyme activity. The enzyme activity of aspartase (U / mL): The enzyme activity standard is that the conversion of 1 μmol of aspartic acid in 1 min is 1 U.

[0053] Determination of aspartic acid content by liquid chromatography: The detector was an ultraviolet detector, the characteristic absorption peak was 205 nm, the chromatographic column was a Karmasil 100 - 5 - NH2 amino column (250 mm × 5 mm), the mobile phase was 50 mmol / L KH2PO4 (containing 0.5% ammonia water, pH = 4.3): acetonitrile = 37:63, the column temperature was 25 °C, the injection volume was 20 μL, the flow rate was 1.3 ml / min, the retention time of fumaric acid was 11.6 min, and the retention time of aspartic acid was 16.4 min.

[0054] Table 2: Comparison of aspartase enzyme activities between the engineered bacteria of aspartase mutants and the starting strain

[0055]

[0056]

[0057] The results of the enzyme activity detection of the wild-type aspartase strain and mutant strains are shown in Table 2. It was found that the catalytic activity was improved after pairwise combined mutations and triple combined mutations compared with single-point mutations. The enzyme activity of the triple combined mutation was increased most significantly, about 13 times higher than that of the wild enzyme.

[0058] Example 5: Study on the thermostatic accelerated storage stability of aspartase and its mutants

[0059] The thermostatic accelerated storage stability of the liquid enzymes of aspartase and its mutants was studied. Accelerated stability tests were carried out at two temperature conditions of 4 °C and -20 °C, and the enzyme activity was detected. The results are as Figure 1 、 Figure 2 shown:

[0060] For the initial enzyme of A-O, the enzyme activity decreased significantly at the initial stage, 3 days, 5 days, 10 days, and 15 days under the storage condition of 4 °C. Compared with the initial stage, it decreased by 93.3% on the 15th day. Similar to A-O, the enzyme activity of A-K decreased by 90% on the 15th day. While for A-G, A-M, A-MK, A-KG, A-MG, and A-MKG, the enzyme activities decreased by 62.2%, 66.3%, 41.9%, 43.5%, 28.4%, and 23.17% respectively after storing at 4 °C for 15 days. Their storage stability was significantly better than that of A-O and A-K.

[0061] For the initial enzyme of A-O, the enzyme activity decreased significantly at the initial stage, 1 month, 2 months, 3 months, and 6 months under the storage condition of -20 °C. Compared with the initial stage, it decreased by 72.2% on the 6th month. The enzyme activity of A-K decreased by 79.6% on the 6th month. While for A-G, A-M, A-MK, A-KG, A-MG, and A-MKG, their storage stability was better at -20 °C. The enzyme activities decreased by 31.3%, 38.4%, 26.3%, 27.2%, 23.2%, and 19.0% respectively on the 6th month.

[0062] Example 6: Biocatalysis of mutants

[0063] Reaction system: 1 L. Prepare a 200 g / L fumaric acid solution, adjust the pH to 8.5 - 8.6 with ammonia water, add 0.2 g of magnesium sulfate, make the volume up to 1 L, react at 38 °C for 1 h, the enzyme dosage is 220 U / g substrate. Detect the contents of fumaric acid and aspartic acid and calculate the conversion efficiency. Finally, the conversion rate of fumaric acid in the reaction system reached more than 99.76% in 1 h.

[0064] Table 3: Comparison of the conversion efficiency between the aspartase mutant engineering bacteria and the starting strain

[0065]

[0066]

[0067] In a 1 L reaction system, after 1 h of conversion, as shown in Table 3, the efficiency of the mutant enzyme in converting fumaric acid is significantly better than that of the wild enzyme, and the conversion rate reaches over 99%.

[0068] Example 7: Applying the crude enzyme solution of the triple mutant A-MKG to a reaction system of more than one ton to convert and generate aspartic acid

[0069] The conversion liquid reaction system is 1000 L: Prepare a 200 g / L fumaric acid solution, adjust the pH to 8.5 - 8.6 with ammonia water, add 0.2 kg of magnesium sulfate, make up the volume to 1000 L, react at 38 °C for 5 - 6 h, the enzyme addition amount is 220 U / g of substrate, detect the contents of fumaric acid and aspartic acid and calculate the conversion efficiency. Finally, the conversion rate of fumaric acid reaches over 99.72% after 5 h in the reaction system.

[0070] Table 4: Comparison of the conversion efficiency results of the crude enzyme solution applied to a reaction system of more than one ton

[0071]

[0072] In a 1000 L reaction system, after 4 h of conversion, as shown in Table 4, the efficiency of the triple mutant A-MKG enzyme in converting fumaric acid reaches over 99%.

[0073] SEQ ID NO:1: Amino acid sequence of aspartase aspA1

[0074] MSNNIRIEEDLLGTREVPADAYYGVHTLRAIENFYISNNKISDIPEFVRGMVMVKKAAA

[0075] MANKELQTIPKSVANAIIAACDEVLNNGKCMDQFPVDVYQGGAGTSVNMNTNEVLA

[0076] NIGLELMGHQKGEYQYLNPNDHVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREG

[0077] FERKAVEFQDILKMGRTQLQDAVPMTLGQEFRAFSILLKEEVKNIQRTAELLLEVNLGA

[0078] TAIGTGLNTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKRLAVK

[0079] MSKICNDLRLLSSGPRAGLNEINLPELQAGSSIMPAKVNPVVPEVVNQVCFKVIGNDTT

[0080] VTMAAEAGQLQLNVMEPVIGQAMFESVHILTNACYNLLEKCINGITANKEVCEGYVY

[0081] NSIGIVTYLNPFIGHHNGDIVGKICAETGKSVREVVLERGLLTEAELDDIFSVQNLMHPAYKAKR*

[0082] SEQ ID NO:2: Amino acid sequence of aspartase mutant

[0083] MSNNIRIEEDLLGTREVPADAYYGVHTLRAIENFYISNNKISDIPEFVRGMVMVKKAAA

[0084] MANKELQTIPKSVANAIIAACDEVLNNGKCHDQFPVDVYQGGAGTSVNMNTNEVLAN

[0085] IGLELMGHQRGEYQYLNPNDHVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREGFE

[0086] RKAVEFQDILKMGRTQLQDAVPMTLGQEFRAFSILLKEEVKNIQRTAELLLEVNLGATA

[0087] IGTGLNTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKRLAVKMS

[0088] KICNDLRLLSSGPRAGLNEINLPELQAGSSIMPAKVNPVVPEVVNQVCFKVIGNDTTVT

[0089] MAAEAGQLQLNVMEPVIGQAMFESVHILTNACYNLLEKCINGITANKEVCEGYVYNSI

[0090] CIVTYLNPFIGHHNGDIVGKICAETGKSVREVVLERGLLTEAELDDIFSVQNLMHPAYKAKR*

[0091] SEQ ID NO:3: aspA1 gene from Escherichia coli K-12

[0092] Atgtcaaacaacattcgtatcgaagaagatctgttgggtaccagggaagttccagctgatgcctactatggtgttcacactctgagagcga

[0093] ttgaaaacttctatatcagcaacaacaaaatcagtgatattcctgaatttgttcgcggtatggtaatggttaaaaaagccgcagctatggcaa

[0094] acaaagagctgcaaaccattcctaaaagtgtagcgaatgccatcattgccgcatgtgatgaagtcctgaacaacggaaaatgcatggatc

[0095] agttcccggtagacgtctaccagggcggcgcaggtacttccgtaaacatgaacaccaacgaagtgctggccaatatcggtctggaactg

[0096] atgggtcaccaaaaaggtgaatatcagtacctgaacccgaacgaccatgttaacaaatgtcagtccactaacgacgcctacccgaccgg

[0097] tttccgtatcgcagtttactcttccctgattaagctggtagatgcgattaaccaactgcgtgaaggctttgaacgtaaagctgtcgaattccag

[0098] gacatcctgaaaatgggtcgtacccagctgcaggacgcagtaccgatgaccctcggtcaggaattccgcgctttcagcatcctgctgaaa

[0099] gaagaagtgaaaaacatccaacgtaccgctgaactgctgctggaagttaaccttggtgcaacagcaatcggtactggtctgaacacgcc

[0100] gaaagagtactctccgctggcagtgaaaaaactggctgaagttactggcttcccatgcgtaccggctgaagacctgatcgaagcgacct

[0101] ctgactgcggcgcttatgttatggttcacggcgcgctgaaacgcctggctgtgaagatgtccaaaatctgtaacgacctgcgcttgctctct

[0102] tcaggcccacgtgccggcctgaacgagatcaacctgccggaactgcaggcgggctcttccatcatgccagctaaagtaaacccggttgt

[0103] tccggaagtggttaaccaggtatgcttcaaagtcatcggtaacgacaccactgttaccatggcagcagaagcaggtcagctgcagttgaa

[0104] cgttatggagccggtcattggccaggccatgttcgaatccgttcacattctgaccaacgcttgctacaacctgctggaaaaatgcattaacg

[0105] gcatcactgctaacaaagaagtgtgcgaaggttacgtttacaactctatcggtatcgttacttacctgaacccgttcatcggtcaccacaac

[0106] ggtgacatcgtgggtaaaatctgtgccgaaaccggtaagagtgtacgtgaagtcgttctggaacgcggtctgttgactgaagcggaactt

[0107] gacgatattttctccgtacagaatctgatgcacccggcttacaaagcaaaacgctaa

[0108] SEQ ID NO:4:Fp:M90H

[0109] cggaaaatgccacgatcagttcccg

[0110] SEQ ID NO:5: Rp: M90H

[0111] cgggaactgatcgtggcattttccg

[0112] SEQ ID NO:6: Fp: K126R

[0113] ggtactgatattcaccacgttggtgacccatcag

[0114] SEQ ID NO:7: Rp: K126R

[0115] ctgatgggtcaccaacgtggtgaatatcagtacc

[0116] SEQ ID NO:8: Fp: G410C

[0117] caactctatctgcatcgttacttacctgaa

[0118] SEQ ID NO:9: Rp: G410C

[0119] ttcaggtaagtaacgatgcagatagagttg

[0120] SEQ ID NO:10: Fp: H26Q

[0121] tatggtgttcaaactctgagagcgat

[0122] SEQ ID NO:11: Rp: H26Q

[0123] tcagagtttgaacaccatagtaggca

Claims

1. An aspartase mutant, characterized in that, The aspartase mutant contains one or more mutations among G410C, M90H or K126R as compared with the amino acid sequence shown in SEQ ID NO:

1.

2. The aspartase mutant according to claim 1, wherein The aspartase mutant contains G410C, M90H, K126R, M90H / K126R / G410C, M90H / G410C, K126R / G410C or M90H / K126R; Preferably, the aspartase mutant contains M90H / K126R / G410C, M90H / G410C, K126R / G410C or M90H / K126R; More preferably, the aspartase mutant contains M90H / K126R / G410C.

3. An isolated nucleic acid encoding the aspartase mutant according to claim 1 or 2.

4. A recombinant expression vector comprising the isolated nucleic acid according to claim 3, and the backbone of the recombinant expression vector is preferably a plasmid of pET-28a, pUC19, pBAD, pKD3, pTrc99a, pBR322, pDG148 or pPIC9.

5. A transformant comprising the isolated nucleic acid according to claim 3 or the recombinant expression vector according to claim 4.

6. The transformant according to claim 5, characterized in that, The host cell used in constructing the transformant is Escherichia coli, Bacillus subtilis and Bacillus licheniformis, and the Escherichia coli is preferably Escherichia coli BL21(DE3).

7. A method for preparing the aspartase mutant according to claim 1 or 2, which comprises culturing the transformant according to claim 5 or 6 to obtain a fermentation product, and obtaining the aspartase mutant from the fermentation product.

8. A method for producing aspartic acid, characterized in that, Produced using the aspartase mutant according to claim 1 or 2 or the transformant according to claim 5 or 6.

9. The method according to claim 8, characterized in that, The method uses fumaric acid as a substrate.

10. Use of the aspartase mutant according to claim 1 or 2, the isolated nucleic acid according to claim 3, the recombinant expression vector according to claim 4 or the transformant according to claim 5 or 6 in the production of aspartic acid.

Images