CN118620879A - L-aspartase mutant and application thereof - Google Patents

Abstract

The invention provides an L-aspartic acid enzyme mutant, which is characterized in that threonine at 221 th position in SEQ ID NO.1 is replaced by glutamine. The invention also provides application of the composition in preparing L-aspartic acid. The specific enzyme activity of the L-aspartic acid enzyme mutant T221Q is 170.6U.mg ^‑1, which is close to that of the wild type; the optimal temperature and the optimal pH are unchanged; the heat stability is significantly improved (after 30min treatment at 50 ℃, the residual enzyme activity of the mutant T221Q is 92.16%, while the residual enzyme activity of the wild type is only 52.9%). The transformation rate of the mutant T221Q single enzyme engineering strain is obviously improved compared with that of the original strain, the transformation rate is improved from 67.1% of the original strain to 93.2% in 2h, the transformation completion time is advanced from 5h to 3h, and the productivity is improved from 47.92 g.L ^‑1·h^‑1 to 79.86 g.L ^‑1·h^‑1.

Description

L-aspartase mutant and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to an L-asparaginase mutant and application thereof.

Background

L-aspartic acid is one of twenty basic amino acids, participates in various metabolic pathways in organisms, and has wide application in the fields of food, medicine, chemical industry and the like. The synthesis method of L-aspartic acid mainly comprises a chemical synthesis method and an enzyme catalysis method, wherein the chemical synthesis method needs to react at high temperature and high pressure, has high equipment requirements, and has difficult separation and purification because the product is raceme DL-aspartic acid. Compared with the prior art, the enzyme catalysis method has the advantages of mild reaction conditions, strong specificity, high product purity, environmental protection and the like, and is a main development direction in the future. The enzyme catalysis method can be classified into a single enzyme method and a double enzyme coupling method, wherein the single enzyme method catalyzes substrate fumaric acid to produce L-aspartic acid by L-aspartase (L-ASPARTASE AMMONIA-lyase, aspA, EC 4.3.1.1); the double enzyme coupling method produces L-aspartic acid from the cheaper substrate maleic acid by tandem connection of maleic acid cis-trans isomerase (MALEATE CIS-trans isomerase, maiA, EC 5.2.1.1) and L-aspartase. L-aspartic acid enzyme is a key enzyme for directly producing L-aspartic acid, and plays a vital role in the production thereof.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an L-aspartase mutant and application thereof in preparing L-aspartic acid.

A first object of the present invention is to provide an L-aspartic acid enzyme mutant in which threonine at position 221 of SEQ ID NO.1 is replaced with glutamine.

A second object of the present invention is to provide a nucleotide sequence encoding the above L-aspartase mutant.

Preferably, the nucleotide sequence is SEQ ID NO.4 in the sequence table.

It is a third object of the present invention to provide a recombinant expression vector comprising the above nucleotide sequence;

preferably, the recombinant expression vector comprises pET-24a (+).

The fourth object of the present invention is a genetically engineered bacterium comprising the recombinant expression vector described above;

Preferably, the genetically engineered bacterium is a recombinant strain obtained by connecting the nucleotide sequences with a vector to obtain a recombinant expression vector, and then introducing the recombinant expression vector into host bacteria for induced expression, wherein the host bacteria comprise escherichia coli, bacillus subtilis or yeast.

The fifth object of the invention is to provide the application of the L-aspartase mutant, nucleotide sequence, recombinant expression vector and genetic engineering bacteria in preparing L-aspartic acid.

Preferably, the application is: the L-aspartic acid is prepared by adding the genetically engineered bacterium of claim 5 into fumaric acid serving as a substrate to perform an enzymatic conversion reaction.

The sixth object of the present invention is to provide a method for preparing L-aspartic acid, wherein fumaric acid is used as a substrate, and the above genetically engineered bacteria are added for enzymatic conversion reaction to prepare L-aspartic acid;

preferably, the genetically engineered bacterium is purified when performing an enzymatic conversion reaction.

Preferably, when the enzyme conversion reaction is carried out, the reaction is carried out at a constant temperature of 35-55 ℃ in a reaction system with the pH value of 6.0-9.0;

Preferably, the enzymatic conversion reaction is carried out in a reaction system having a pH of 8.0 at a constant temperature of 37 ℃;

As a further preference, the reaction time is from 2 to 5 hours.

Preferably, the final concentration of fumaric acid is 1.8 mol.L ^-1.

The specific enzyme activity of the L-aspartic acid enzyme mutant T221Q is 170.6U.mg ^-1, which is close to that of the wild type; the optimal temperature and the optimal pH are unchanged; the heat stability is significantly improved (after 30min treatment at 50 ℃, the residual enzyme activity of the mutant T221Q is 92.16%, while the residual enzyme activity of the wild type is only 52.9%). The transformation rate of the mutant T221Q single enzyme engineering strain is obviously improved compared with that of the original strain, the transformation rate is improved from 67.1% of the original strain to 93.2% in 2h, the transformation completion time is advanced from 5h to 3h, and the productivity is improved from 47.92 g.L ^-1·h^-1 to 79.86 g.L ^-1·h^-1.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:

FIG. 1 is a map of plasmid pET-24a (+) -EcAspA.

FIG. 2 is a SDS-PAGE map of wild type and mutant T221Q.

FIG. 3 is a graph showing the enzymatic activity of wild type and mutant T221Q at different temperatures.

FIG. 4 is a graph showing the enzymatic activity of wild type and mutant T221Q at different pH.

FIG. 5 is a graph showing the thermal stability of wild-type and mutant T221Q incubated at temperature for half an hour.

FIG. 6 shows whole cell catalysis of wild type and mutant T221Q single enzyme engineered strains.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

LB medium: peptone 10 g.L ^-1, yeast extract 5 g.L ^-1,NaCl 10g·L^-1.

2 XYT fermentation medium: peptone 10 g.L ^-1, yeast extract 5 g.L ^-1、NaCl 10g·L^-1.

L-aspartic acid enzyme Activity definition: the amount of enzyme required to convert substrate fumaric acid to 1mmol of product L-aspartic acid per minute at 37℃and pH 7.0 is one enzyme activity unit U. The unit of enzyme activity per mg of protein is defined as specific enzyme activity.

Method for measuring enzymatic properties of L-aspartic acid enzyme:

(1) The enzyme activity determination method comprises the following steps: 1mL of a reaction system containing 0.01 mg/mL ^-1 of pure enzyme, 500 mmoL/L ^-1 of fumaric acid substrate and 50 mmoL/L ^-1 of Na ₂HPO₄-KH₂PO₄ buffer. Reacting at 37 ℃ for 10min, inactivating in a metal bath at 100 ℃ for 15min, centrifuging at 12000 r.min ^-1 min by a centrifuge, and taking the supernatant to be diluted appropriately for subsequent detection.

(2) The optimal temperature detection method comprises the following steps: the enzyme activities were measured at different temperatures (35 ℃ C. -55 ℃ C.). The highest enzyme activity was defined as 100% and the change in enzyme activity at different temperatures was analyzed.

(3) The optimal pH detection method comprises the following steps: the enzyme activities were measured at different pH (6.0-9.0). The highest enzyme activity was defined as 100% and the change in enzyme activity at different pH conditions was analyzed.

(4) The temperature stability detection method comprises the following steps: the purified enzyme was incubated in a metal bath at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃at 65℃at 70℃at 75℃for 30min, then cooled on ice for 10min, and the enzyme activity was measured at 37℃and the highest enzyme activity was defined as 100%, and the temperature stability was analyzed.

(5) Pretreatment of amino acid samples: diluting a sample to be detected by deionized water to the concentration of 0.5 mmol.L ^-1-3mmol·L^-1, taking 500 mu L of diluted sample into a 2mL centrifuge tube, and sequentially adding 250 mu L of triethylamine: acetonitrile=97: solution 3 and 250 μ LPITC: acetonitrile = 1:83, fully and uniformly mixing the solution, and carrying out light-shielding reaction for 50min. Add 750. Mu.L of n-hexane solution to terminate derivatization, mix well, and stand for delamination. The lower layer solution is sucked by a syringe with a needle tube, filtered by a 0.2 mu organic filter membrane to remove impurities, placed in a 2mL liquid phase vial, and covered by a bottle cap to avoid volatilization of the organic solution.

(6) Amino acid detection method: detection was performed by HPLC, column Diamonsil μm C (2), 250×4.6mm; column temperature 40 ℃; double mobile phase, mobile phase A is 80% acetonitrile-water solution by volume ratio, mobile phase B is 0.1 mol.L ^-1 sodium acetate: acetonitrile volume ratio = 97: 3; the detection time is 35min; in the process of gradient of 0 min-30 min-35 min, the proportion of the mobile phase A is linearly changed according to the volume percentage of 5-30-5%, and the proportion of the mobile phase B is linearly changed according to the volume percentage of 95-70-95%; the flow rate is 0.6 mL.min ^-1; the detection wavelength is 254nm.

EXAMPLE 1 construction and purification of L-aspartase mutant T221Q

The synthesized plasmid pET-24a (+) -EcAspA (EcAspA has a nucleotide sequence of SEQ ID NO.2 in a sequence table) is used as a template, and the primers P1 and P2 are shown in the table 1. Full plasmid PCR was performed and the PCR system and conditions are shown in Table 2 and Table 3.

FIG. 1 is a map of plasmid pET-24a (+) -EcAspA.

TABLE 1 primers

TABLE 2PCR reaction System

Template plasmid	1μL
		Upper/lower primers	1 Mu L each
2X PrimeStarMixDNA polymerase	25μL
		ddH2O	22μL
Totalizing	50μL

TABLE 3PCR reaction conditions

And (3) performing agarose electrophoresis verification on the PCR product, then performing digestion and purification, transforming the purified PCR plasmid into E.coli JM109 competent, and sequencing the single colony of the LB solid plate after transformation culture.

The correct sequencing plasmid pET-24a (+) -EcAspA/T221Q was transformed into competent E.coli BL21 (DE 3) and cultured overnight. Adding 1/millkanamycin by weight percent into a 5mL LB culture medium test tube, picking a single colony with good growth vigor at the center of a flat plate, and culturing for 6h in the test tube by a constant temperature shaking table 200 r.min ^-1 at 37 ℃.200mL 2YT medium shake flask is added with 1/millkanamycin by weight percent, 2% (v/v) test tube bacterial liquid is inoculated, and a constant temperature shaking table 200 r.min ^-1 at 37 ℃ is used for culturing until OD ₆₀₀ =about 0.8. Adding inducer IPTG with final concentration of 0.2 mmol.L ^-1, and continuously culturing at 24 ℃ for 14h in a shaking table 200 r.min ^-1.

Freezing at 4deg.C, centrifuging at 6000 r.min ^-1 for 10min, collecting thallus, suspending with Binding buffer, controlling temperature, ultrasonic crushing for 30min until the liquid is clear and transparent, centrifuging at 4deg.C, 13000 r.min ^-1 for 30min, collecting supernatant, and filtering with 0.22 μm filter membrane for low temperature storage.

The 1mL His Trap HP column was rinsed with deionized water, and then 5mL of 0.1 mol.L ^-1NiSO₄ was injected to ensure adequate Ni ion binding. The specific steps of purifying the protein by using an AKTA purifier are as follows: the 1mL mLHis Trap HP column was washed with 10mL Washingbuffer and the 1mL His Trap HP column was equilibrated with 20mL Binding buffer to baseline plateau. A proper amount of sample is taken and loaded, nonspecifically adsorbed proteins are washed off by using a Binding buffer of 20mL until the baseline is stable, the proteins are eluted linearly by using 27mLWashingbuffer, and the elution peak is collected for later study.

And (3) dialysis: 50 mmol.L ^-1Na₂HPO₄-KH₂PO₄ buffer, pH=8.0, dialyzed overnight.

SDS-PAGE analysis was performed on the bacterial liquid and the pure enzyme, and the results are shown in FIG. 2.

The specific enzyme activity of L-aspartic acid enzyme mutant T221Q (abbreviated as "mutant T221Q") and the specific enzyme activity of wild-type L-aspartic acid enzyme were measured, respectively, to obtain that the specific enzyme activity of L-aspartic acid enzyme mutant T221Q was 170.6U.mg ^-1, and the specific enzyme activity of wild-type L-aspartic acid enzyme was 168.2U.mg ^-1, which were relatively close to each other.

FIG. 2 is a SDS-PAGE map of wild type and mutant T221Q.

The amino acid sequence of the wild-type L-aspartic acid enzyme is as follows (SEQ ID NO.1 of the sequence Listing):

MSNNIRIEEDLLGTREVPADAYYGVHTLRAIVNFYISNNKISDIPEFVRGMVMVKKAAAMANKELQTIPKSVANAIIAACDEVLNNGKCMDQFPVDVYQGGAGTSVNMNTNEVLANIGLELMGHQKGEYQYLNPNDHVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREGFERKAVEFQDILKMGRTQLQDAVPMTLGQEFRAFSILLKEEVKNIQRTAELLLEVNLGATAIGTGLNTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKRLAVKMSKICNDLRLLSSGPRAGLNEINLPELQAGSSIMPAKVNPVVPEVVNQVCFKVIGNDTTVTMAAEAGQLQLNVMEPVIGQAMFESVHILTNACYNLLEKCINGITANKEVCEGYVYNSIGIVTYLNPFIGHHNGDIVGKICAETGKSVREVVLERGLLTEAELDDIFSVQNLMHPAYKAKRYTDESEQ

The wild-type L-aspartase encoding nucleotide sequence is as follows (SEQ ID NO.2 of the sequence Listing):

atgtcaaacaacattcgtatcgaagaagatctgttgggtaccagggaagttccagctgatgcctactatggtgttcacactctgagagcgattgaaaacttctatatcagcaacaacaaaatcagtgatattcctgaatttgttcgcggtatggtaatggttaaaaaagccgcagctatggcaaacaaagagctgcaaaccattcctaaaagtgtagcgaatgccatcattgccgcatgtgatgaagtcctgaacaacggaaaatgcatggatcagttcccggtagacgtctaccagggcggcgcaggtacttccgtaaacatgaacaccaacgaagtgctggccaatatcggtctggaactgatgggtcaccaaaaaggtgaatatcagtacctgaacccgaacgaccatgttaacaaatgtcagtccactaacgacgcctacccgaccggtttccgtatcgcagtttactcttccctgattaagctggtagatgcgattaaccaactgcgtgaaggctttgaacgtaaagctgtcgaattccaggacatcctgaaaatgggtcgtacccagctgcaggacgcagtaccgatgaccctcggtcaggaattccgcgctttcagcatcctgctgaaagaagaagtgaaaaacatccaacgtaccgctgaactgctgctggaagttaaccttggtgcaacagcaatcggtactggtctgaacacgccgaaagagtactctccgctggcagtgaaaaaactggctgaagttactggcttcccatgcgtaccggctgaagacctgatcgaagcgacctctgactgcggcgcttatgttatggttcacggcgcgctgaaacgcctggctgtgaagatgtccaaaatctgtaacgacctgcgcttgctctcttcaggcccacgtgccggcctgaacgagatcaacctgccggaactgcaggcgggctcttccatcatgccagctaaagtaaacccggttgttccggaagtggttaaccaggtatgcttcaaagtcatcggtaacgacaccactgttaccatggcagcagaagcaggtcagctgcagttgaacgttatggagccggtcattggccaggccatgttcgaatccgttcacattctgaccaacgcttgctacaacctgctggaaaaatgcattaacggcatcactgctaacaaagaagtgtgcgaaggttacgtttacaactctatcggtatcgttacttacctgaacccgttcatcggtcaccacaacggtgacatcgtgggtaaaatctgtgccgaaaccggtaagagtgtacgtgaagtcgttctggaacgcggtctgttgactgaagcggaacttgacgatattttctccgtacagaatctgatgcacccggcttacaaagcaaaacgctatactgatgaaagcgaacagtaa

The amino acid sequence of the L-aspartic acid enzyme mutant is as follows (SEQ ID NO.3 of the sequence Listing):

MSNNIRIEEDLLGTREVPADAYYGVHTLRAIVNFYISNNKISDIPEFVRGMVMVKKAAAMANKELQTIPKSVANAIIAACDEVLNNGKCMDQFPVDVYQGGAGTSVNMNTNEVLANIGLELMGHQKGEYQYLNPNDHVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREGFERKAVEFQDILKMGRTQLQDAVPMTLGQEFRAFSILLKEEVKNIQRQAELLLEVNLGATAIGTGLNTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKRLAVKMSKICNDLRLLSSGPRAGLNEINLPELQAGSSIMPAKVNPVVPEVVNQVCFKVIGNDTTVTMAAEAGQLQLNVMEPVIGQAMFESVHILTNACYNLLEKCINGITANKEVCEGYVYNSIGIVTYLNPFIGHHNGDIVGKICAETGKSVREVVLERGLLTEAELDDIFSVQNLMHPAYKAKRYTDESEQ

The bolded and underlined portions are mutation sites.

That is, based on the amino acid sequence of the wild-type L-aspartic acid enzyme, only the 221 th amino acid is mutated, and threonine T is mutated into glutamine Q.

The L-aspartic acid enzyme mutant T221Q coding nucleotide sequence is as follows (SEQ ID NO.4 of the sequence Listing):

atgtcaaacaacattcgtatcgaagaagatctgttgggtaccagggaagttccagctgatgcctactatggtgttcacactctgagagcgattgaaaacttctatatcagcaacaacaaaatcagtgatattcctgaatttgttcgcggtatggtaatggttaaaaaagccgcagctatggcaaacaaagagctgcaaaccattcctaaaagtgtagcgaatgccatcattgccgcatgtgatgaagtcctgaacaacggaaaatgcatggatcagttcccggtagacgtctaccagggcggcgcaggtacttccgtaaacatgaacaccaacgaagtgctggccaatatcggtctggaactgatgggtcaccaaaaaggtgaatatcagtacctgaacccgaacgaccatgttaacaaatgtcagtccactaacgacgcctacccgaccggtttccgtatcgcagtttactcttccctgattaagctggtagatgcgattaaccaactgcgtgaaggctttgaacgtaaagctgtcgaattccaggacatcctgaaaatgggtcgtacccagctgcaggacgcagtaccgatgaccctcggtcaggaattccgcgctttcagcatcctgctgaaagaagaagtgaaaaacatccaacgtcaagctgaactgctgctggaagttaaccttggtgcaacagcaatcggtactggtctgaacacgccgaaagagtactctccgctggcagtgaaaaaactggctgaagttactggcttcccatgcgtaccggctgaagacctgatcgaagcgacctctgactgcggcgcttatgttatggttcacggcgcgctgaaacgcctggctgtgaagatgtccaaaatctgtaacgacctgcgcttgctctcttcaggcccacgtgccggcctgaacgagatcaacctgccggaactgcaggcgggctcttccatcatgccagctaaagtaaacccggttgttccggaagtggttaaccaggtatgcttcaaagtcatcggtaacgacaccactgttaccatggcagcagaagcaggtcagctgcagttgaacgttatggagccggtcattggccaggccatgttcgaatccgttcacattctgaccaacgcttgctacaacctgctggaaaaatgcattaacggcatcactgctaacaaagaagtgtgcgaaggttacgtttacaactctatcggtatcgttacttacctgaacccgttcatcggtcaccacaacggtgacatcgtgggtaaaatctgtgccgaaaccggtaagagtgtacgtgaagtcgttctggaacgcggtctgttgactgaagcggaacttgacgatattttctccgtacagaatctgatgcacccggcttacaaagcaaaacgctatactgatgaaagcgaacagtaa

The bolded and underlined portions are mutation sites.

That is, based on the nucleotide sequence of wild-type L-aspartic acid enzyme, only the 661-663 th base is mutated from acc to caa.

Example 2 optimum temperature measurement

The optimal temperature was measured using the mutant T221Q and the wild type in example 1, respectively, and 1mL of the reaction system was prepared, which contained 0.01 mg/mL ^-1 of pure enzyme, 500 mmoL/L ^-1 of fumaric acid substrate, and 50 mmoL/L ^-1 of Na ₂HPO₄-KH₂PO₄ buffer. Respectively reacting at 35deg.C, 40deg.C, 45deg.C, 50deg.C and 55deg.C for 10min, inactivating in metal bath at 100deg.C for 15min, centrifuging 12000 r.min ^-1 min, and diluting the supernatant.

As shown in FIG. 3, the optimal temperature of the mutant T221Q and the wild type is 45 ℃, and the relative enzyme activity of the mutant T221Q is maintained above 90% at 35-55 ℃.

FIG. 3 is a graph showing the enzymatic activity of wild type and mutant T221Q at different temperatures.

Example 3 determination of optimum pH

The optimal pH was measured using the mutant T221Q and the wild type in example 1, respectively, and 1mL of the reaction system was prepared, which contained 0.01 mg/mL ^-1 of pure enzyme, 500 mmoL/L ^-1 of fumaric acid substrate, and 50 mmoL/L ^-1 of Na ₂HPO₄-KH₂PO₄ buffer. Respectively reacting for 10min under the conditions of buffer solution pH 6.0, 7.0, 8.0 and 9.0, inactivating for 15min in a metal bath at 100 ℃, centrifuging for 5min in a centrifuge 12000 r.min ^-1, and taking supernatant to dilute properly for detection.

As shown in FIG. 4, the optimal pH of the mutant T221Q and the wild type was 8.0, and the relative enzyme activities of the mutant T221Q were 90% or more in the pH range of 6.0-9.0.

FIG. 4 is a graph showing the enzymatic activity of wild type and mutant T221Q at different pH.

Example 4 temperature stability determination

The wild-type and mutant T221Q pure enzymes were incubated in a metal bath at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃at 65℃at 70℃at 75℃for 30min, respectively, and then cooled on ice for 10min. 1mL of the reaction system contains 0.01 mg/mL ^-1 of pure enzyme, 500 mmoL/L ^-1 of fumaric acid substrate and 50 mmoL/L ^-1 of Na ₂HPO₄-KH₂PO₄ buffer. Respectively reacting at 37deg.C for 10min, inactivating at 100deg.C in metal bath for 15min, centrifuging at 12000 r.min ^-1 min, and diluting supernatant.

As a result, as shown in FIG. 5, the mutant T221Q had a thermostability superior to that of the wild type, and after 30 minutes of treatment at 50℃the residual enzyme activity of the mutant T221Q was 92.16%, whereas the residual enzyme activity of the wild type was only 52.9%.

FIG. 5 is a graph showing the thermal stability of wild-type and mutant T221Q incubated at different temperatures for half an hour.

EXAMPLE 5 construction of Mono-enzyme engineering Strain and L-aspartic acid production

The wild type and the mutant plasmids of example 1 were transformed into competent E.coli BL21 (DE 3), respectively, and incubated overnight in an incubator at 37 ℃. Adding 1/millkanamycin by weight percent into a 5mL LB culture medium test tube, picking a single colony with good growth vigor at the center of a flat plate, and culturing for 6h in the test tube by a constant temperature shaking table 200 r.min ^-1 at 37 ℃.50mL 2YT medium shake flask is added with 1/millkanamycin by weight percent, 2% (v/v) of test tube bacterial liquid is inoculated, and the culture is carried out by a constant temperature shaking table 200 r.min ^-1 at 37 ℃ until OD ₆₀₀ =about 0.8. Adding inducer IPTG with final concentration of 0.2 mmol.L ^-1, and continuously culturing at 24 ℃ for 14h in a shaking table 200 r.min ^-1.

Transferring the induced expression bacterial liquid into a 50mL centrifuge tube, and balancing with deionized water. Centrifuging at 4deg.C for 5min at 6000 r.min ^-1 min, discarding supernatant, and re-suspending the precipitate with deionized water. The reaction solution was prepared from the resuspended strain, 2 mol.L ^-1 of fumaric acid solution (pH 8.0 was adjusted with ammonia water) and deionized water, 30mL of the reaction system was used, the cell OD ₆₀₀ =0.25, and the substrate fumaric acid final concentration was 1.8 mol.L ^-1. The reaction is carried out by a constant temperature shaking table at 37 ℃ for 200 r.min ^-1, 500 mu L of sample is taken per hour and placed in a centrifuge tube, metal bath is inactivated at 100 ℃ for 15min, centrifugal machine 13000 r.min ^-1 is used for centrifugation for 5min, and supernatant is taken and properly diluted for detection.

As shown in FIG. 6, the transformation rate of the mutant strain is obviously improved, the transformation rate is 93.2% in 2h and is higher than 67.1% of that of the original strain, the transformation completion time is advanced from 5h to 3h, and the productivity is improved from 47.92 g.L ^-1·h^-1 to 79.86 g.L ^-1·h^-1.

FIG. 6 shows whole cell catalysis of wild type and mutant T221Q single enzyme engineered strains.

Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. An L-aspartase mutant characterized in that: the threonine at position 221 in SEQ ID NO.1 is replaced by glutamine.

2. A nucleotide sequence encoding the mutant L-aspartic acid enzyme according to claim 1.

3. The nucleotide sequence according to claim 2, characterized in that: the nucleotide sequence is SEQ ID NO.4 in the sequence table.

4. A recombinant expression vector comprising the nucleotide sequence of claim 2 or 3;

preferably, the recombinant expression vector comprises pET-24a (+).

5. A genetically engineered bacterium comprising the recombinant expression vector of claim 4;

preferably, the genetically engineered bacterium is a recombinant strain obtained by connecting the nucleotide sequence of claim 2 or 3 with a vector to obtain a recombinant expression vector, and then introducing the recombinant expression vector into host bacteria for induction expression, wherein the host bacteria comprise escherichia coli, bacillus subtilis or yeast.

6. Use of the mutant L-aspartase of claim 1, the nucleotide sequence of claim 2 or 3, the recombinant expression vector of claim 4, the genetically engineered bacterium of claim 5 for preparing L-aspartic acid.

7. The use according to claim 6, characterized in that: the application is as follows: the L-aspartic acid is prepared by adding the genetically engineered bacterium of claim 5 into fumaric acid serving as a substrate to perform an enzymatic conversion reaction.

8. A preparation method of L-aspartic acid is characterized in that: adding the genetically engineered bacterium of claim 5 into fumaric acid serving as a substrate to perform an enzymatic conversion reaction to prepare L-aspartic acid;

preferably, the genetically engineered bacterium is purified when performing an enzymatic conversion reaction.

9. The method for producing L-aspartic acid according to claim 8, wherein: in the enzyme conversion reaction, in a reaction system with the pH value of 6.0-9.0, the reaction is carried out at the constant temperature of 35-55 ℃;

Preferably, the enzymatic conversion reaction is carried out in a reaction system having a pH of 8.0 at a constant temperature of 37 ℃;

As a further preference, the reaction time is from 2 to 5 hours.

10. The method for producing L-aspartic acid according to claim 8 or 9, wherein: the final concentration of fumaric acid is 1.8 mol.L ^-1.