Methyl aspartate lyase ecmal mutant, preparation method and application thereof

Abstract

The application discloses a kind of methyl aspartate lyase EcMAL mutant and its preparation method and application, belong to enzyme engineering technical field.The methyl aspartate lyase EcMAL mutant is any one of mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3 and mutant EcMAL-M4.The application is obtained methyl aspartate lyase EcMAL mutant by the mutation of the 329th, 331st, 361st and 365th amino acid sites of methyl aspartate lyase combined with high-throughput screening method.The mutant changes the previous recombinant enzyme only L-aspartic acid production status, so that it is possible to produce D-aspartic acid with methyl aspartate lyase as biological catalyst, and it is expected to be used in pharmaceutical industry.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a methylaspartate lyase EcMAL mutant, its preparation method, and its application. Background Technology

[0002] Chirality is one of the essential properties widely present in nature, and chiral drugs have important applications in drug synthesis and development. The various amino acids that make up the human body (except glycine) also have mirror-image enantiomers, L-type and D-type. Natural L-amino acids form the basic backbone of proteins, while D-amino acids, due to their unique structures, play important roles in medicine, food, and other fields. D-aspartic acid (D-Asp) is a type of D-amino acid and is commonly used as a precursor and intermediate in drug synthesis. Examples include apucillin, the first amino acid-based penicillin injection used clinically; and D-aspartate-β-hydroxylamine (DAH), a drug synthesized using D-Asp as a precursor for treating viral infections and anti-tumor purposes. Furthermore, because D-amino acids are difficult to degrade in vivo, they are less likely to induce corresponding drug resistance. This characteristic makes them highly effective in the synthesis of enzyme inhibitors, and replacing L-amino acids in peptide drugs can significantly extend the half-life of peptide drugs and reduce side effects. In the food industry, D-Asp is also widely used as a substitute for preservatives and flavor modifiers.

[0003] Foreign countries, especially Japan, have long been at the forefront of D-aspartic acid production technology, while domestic reports are scarce. Current methods for preparing D-aspartic acid mainly include physical, chemical, and biological methods. Physical methods primarily obtain the pure product through crystallization. Chemical methods are divided into asymmetric synthesis and resolution. However, both physical and chemical methods suffer from complex processes, lengthy steps, and substandard yields and purity, hindering large-scale application. Biological methods are divided into biological resolution and asymmetric synthesis. Current technologies commonly use biological methods to prepare L-aspartate-α-decarboxylase, L-aspartate-β-decarboxylase, and aspartate enzymes; however, the biological resolution reaction produces byproducts, affecting the yield of D-aspartic acid. Asymmetric synthesis requires the participation of multiple enzymes, and currently, the yield is relatively low, lacking industrial application value. Furthermore, current research has focused on using natural methylaspartate lyases to prepare L-aspartic acid; there are no reports of D-aspartic acid production. Summary of the Invention

[0004] The primary objective of this application is to provide a methylaspartate lyase EcMAL mutant.

[0005] Another objective of this application is to provide a method for preparing the above-mentioned methylaspartate lyase EcMAL mutant.

[0006] Another object of this application is to provide the use of the above-mentioned methylaspartate lyase EcMAL mutant.

[0007] The final objective of this application is to provide a method for preparing D-aspartic acid using a methylaspartate lyase EcMAL mutant.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A methylaspartate lyase EcMAL mutant, which is any one of mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3 and mutant EcMAL-M4.

[0010] The mutant EcMAL-M1 is formed by mutating glutamine at position 329 to aspartic acid, lysine at position 331 to glycine, cysteine ​​at position 361 to histidine, and aspartic acid at position 365 to aspartic acid in the methyl aspartate lyase shown in SEQ ID NO:1.

[0011] The mutant EcMAL-M2 is formed by mutating glutamine at position 329 of the methylaspartate lyase as shown in SEQ ID NO:1 to arginine, lysine at position 331 to isoleucine, cysteine ​​at position 361 to arginine, and aspartic acid at position 365 to leucine.

[0012] The mutant EcMAL-M3 is formed by mutating glutamine at position 329 to histidine, lysine at position 331 to serine, cysteine ​​at position 361 to cysteine, and aspartic acid at position 365 to serine in the amino acid sequence of methylaspartate lyase as shown in SEQ ID NO:1.

[0013] The mutant EcMAL-M4 is formed by mutating glutamine at position 329 of the methylaspartate lyase shown in SEQ ID NO:1 to valine, lysine at position 331 to cysteine, cysteine ​​at position 361 to cysteine, and aspartic acid at position 365 to leucine.

[0014] The amino acid sequence of the mutant EcMAL-M1 is shown in SEQ ID NO:3;

[0015] The amino acid sequence of the mutant EcMAL-M2 is shown in SEQ ID NO:5;

[0016] The amino acid sequence of the mutant EcMAL-M3 is shown in SEQ ID NO:7;

[0017] The amino acid sequence of the mutant EcMAL-M4 is shown in SEQ ID NO:9.

[0018] The nucleotide sequence encoding the mutant EcMAL-M1 gene is shown in SEQ ID NO:4;

[0019] The nucleotide sequence encoding the mutant EcMAL-M2 gene is shown in SEQ ID NO:6;

[0020] The nucleotide sequence encoding the mutant EcMAL-M3 gene is shown in SEQ ID NO:8;

[0021] The nucleotide sequence encoding the mutant EcMAL-M4 gene is shown in SEQ ID NO:10.

[0022] The methylaspartate lyase described is derived from Escherichia coli O157:H7.

[0023] A method for preparing a methylaspartate lyase EcMAL mutant includes the following steps:

[0024] S1. The EcMAL enzyme gene is ligated into a plasmid to obtain a recombinant plasmid;

[0025] S2. Design mutation primers, use the mutation primers and the recombinant plasmid as a template for PCR amplification, and then use the PCR amplification product as a large primer for full plasmid amplification to obtain the mutation product. Transform the mutation product into the host cell to obtain the mutant library.

[0026] S3. High-throughput screening of the mutant library yielded the EcMAL mutant of methylaspartate lyase.

[0027] In step S1, the nucleotide sequence of the EcMAL enzyme gene is as shown in SEQ ID NO.2.

[0028] In step S1, the plasmid is preferably any one of PET-32a(+), PET-28a, PET-22b and PET-21a.

[0029] In step S2, the mutation primers are 329-331-F and 361-365-R:

[0030] 329-331-F: 5'-TGTCACATGTGDTATCNDTACCCGG-3';

[0031] 361-365-R: 5'-GGCACTGACAHNAGTTTCATTAHNGGTGCCG-3';

[0032] NDT represents any one of the twelve amino acids (R / N / D / C / G / H / I / L / F / S / Y / V).

[0033] In step S2, the large primer is:

[0034] Large primer F:

[0035] 5'-TGTCACATGGTGNDTATCNDTACCCCGGATCTGGGCAGTATTCACAACATCGTCGATGCGGTTCTTTACTGCAACAGCCACAGCATGGAAGCGTACCAGGGCGGCACCNDTAATGAAACTNDTGTCAGTGCC-3';

[0036] Large primer R:

[0037] 5'-GGCACTGACAHNAGTTTCATTAHNGGTGCCGCCCTGGTACGCTTCCATGCTGTGGCTGTTGCAGTAAAGAACCGCATCGACGATGTTGTGAATACTGCCCAGATCCGGGGTAHNGATAHNCACCATGTGACA-3'.

[0038] In step S2, the host cell is preferably any one of E. coli BL21(DE3), E. coli BL21(DE3)pLysS, and Rosetta(DE3).

[0039] This invention utilizes the high-throughput screening capability of D-aspartic acid oxidase (D-ASPO) to specifically oxidize D-aspartic acid to iminosuccinic acid and generate the byproduct H2O2, while horseradish peroxidase (HRP) can oxidize the chromogenic agent 3',3-diaminobenzidine (DAB) to a reddish-brown compound in the presence of H2O2.

[0040] In step S3, the preferred method for high-throughput screening of the mutant library to obtain the EcMAL methylaspartate lyase mutant is as follows: the mutant in the mutant library is coated on a culture medium with a nylon membrane attached and cultured and induced to obtain an induced nylon membrane with the mutant; the induced nylon membrane with the mutant is subjected to freeze-thaw treatment to obtain a freeze-thawed nylon membrane with the mutant; the freeze-thawed nylon membrane with the mutant is placed on filter paper containing a mixture and allowed to stand; the appearance of a reddish-brown single colony is the EcMAL methylaspartate lyase mutant; wherein: the mixture is a mixture containing horseradish peroxidase and 3',3-diaminobenzidine (DAB).

[0041] The mixture is preferably composed of the following components: 500mM Tris-HCl, 20mM MgCl2, 500mM NH4Cl, 10mM fumaric acid, 0.1mg / mL HRP, 0.1mM DAB and 0.5mg / mL D-ASPO crude enzyme solution.

[0042] The crude D-ASPO enzyme solution was prepared by the following method: the D-ASPO gene was introduced into Escherichia coli for induced expression and isolation, thus obtaining the crude D-ASPO enzyme solution.

[0043] The gene sequence of D-ASPO is shown in SEQ ID NO.11.

[0044] The filter paper containing the mixture is obtained by immersing the filter paper in the mixture.

[0045] The preferred induction conditions are: induction at 25-35℃ for 3-8 hours; more preferably: induction at 30℃ for 4 hours.

[0046] The preferred static incubation conditions are: static incubation at 20–40°C for at least 30 minutes; more preferably: static incubation at 30°C for 30 minutes.

[0047] The application of the EcMAL mutant of methylaspartate lyase in the preparation of D-aspartic acid.

[0048] A method for preparing D-aspartic acid using the EcMAL mutant of methylaspartate lyase includes the following steps:

[0049] The mutant methylaspartate lyase EcMAL was induced, cultured, isolated, and purified to obtain the mutant enzyme EcMAL. The mutant enzyme EcMAL was then added to the reaction solution to carry out the reaction, thus obtaining D-aspartic acid.

[0050] The reaction solution is preferably composed of the following components: 500mM Tris-HCl, 20mM MgCl2, 500mM NH4Cl, 10mM fumaric acid, pH 8.5.

[0051] The preferred reaction conditions are: 20–40°C, 100–300 rpm for 10–30 h; more preferably: 30°C, 200 rpm for 20 h.

[0052] Compared with the prior art, this application has the following beneficial effects:

[0053] This invention utilizes a high-throughput screening method to simultaneously mutate amino acid sites 329, 331, 361, and 365 of methylaspartate lyase, resulting in the EcMAL mutant. This mutant changes the previous situation where recombinant enzymes only produced L-aspartate, making it possible to produce D-aspartate using methylaspartate lyase as a biocatalyst, which holds promise for applications in the pharmaceutical industry. Attached Figure Description

[0054] Figure 1 This is a graph showing the results of liquid chromatography using a mixed standard of L-aspartic acid and D-aspartic acid.

[0055] Figure 2 This is a liquid phase diagram showing the L-aspartic acid and D-aspartic acid produced by the methylaspartate lyase EcMAL produced by the recombinant strain E.coli BL21(DE3) / pET-32a-mal(EcMAL) after the catalytic reaction with fumaric acid as a substrate.

[0056] Figure 3 This is a liquid phase diagram of L-aspartic acid and D-aspartic acid produced by the EcMAL mutant generated from the EcMAL-M1 mutant after the catalytic reaction with fumaric acid as the substrate.

[0057] Figure 4 This is a liquid phase diagram of L-aspartic acid and D-aspartic acid produced by the EcMAL mutant generated from the EcMAL-M2 mutant after the catalytic reaction with fumaric acid as the substrate.

[0058] Figure 5 This is a liquid phase diagram of L-aspartic acid and D-aspartic acid produced by the EcMAL mutant generated from the EcMAL-M3 mutant after the catalytic reaction with fumaric acid as the substrate.

[0059] Figure 6 This is a liquid phase diagram of L-aspartic acid and D-aspartic acid produced by the EcMAL mutant generated from the EcMAL-M4 mutant after the catalytic reaction with fumaric acid as the substrate. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0061] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the reagents and raw materials used in this invention are commercially available.

[0062] Example 1: Construction of recombinant bacteria containing EcMAL

[0063] 1. Using the E. coli O157:H7 genome as a template, and mal-F and mal-R as primers:

[0064] mal-F:5'-GCTGATATCGGATCC GAATTC ATGAAAATAAAACAGGCTCTGTTCAC-3';

[0065] mal-R: 5'-GTGGTGGTGGTGGTG CTCGAG TTAATCCTTAGCCTGCAACAGCG-3'.

[0066] The *mal* gene in *E. coli* O157:H7 was amplified, and EcoRI and XhoI restriction endonuclease sites (underlined) were introduced at its 5' and 3' ends, respectively. The PCR reaction mixture (50 μL total volume) consisted of: 25 μL 2×PrimeSTARMax Premix, 1 μL template DNA, 1 μL each of forward and reverse primers, and 22 μL sterile water. The PCR conditions were: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, and 72℃ extension for 80 s, for 30 cycles; and a final extension at 72℃ for 10 min. The PCR amplification product *mal* was obtained. The PCR amplification product was verified by 1% agarose gel electrophoresis, and the PCR product was recovered using a PCR product recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The plasmid pET-32a(+) was simultaneously digested with restriction endonucleases EcoRI and XhoI at 37℃ for 4 h. The reaction system is shown in Table 1. After digestion, the digestion products were subjected to agarose gel electrophoresis, and the linear plasmid was recovered by gel extraction using a gel extraction kit. The recovered linear plasmid and PCR products were then processed at a molar ratio of 1:3 (plasmid:target gene). Homologous recombination was performed using the Ultra One Step Cloning Kit (purchased from Novizan Biotechnology Co., Ltd.) to obtain the recombinant plasmid pET-32a-mal. The recombinant plasmid was then transformed into *E. coli* BL21(DE3) competent cells via heat excitation to obtain the recombinant strain *E. coli* BL21(DE3) / pET-32a-mal (EcMAL).

[0067] Table 1:

[0068]

[0069]

[0070] Example 2: Construction, screening, and validation of active mutant libraries

[0071] (1) Construction of mutant libraries

[0072] Based on the amino acid sequence of the mal gene, homology modeling was performed in SWISS-MODEL (https: / / swissmodel.expasy.org / ) to obtain the three-dimensional structure of EcMAL. Using AutoDock Vina software, fumarate was molecularly docked with the three-dimensional structure of EcMAL to identify key amino acid sites (positions 329, 331, and 361). CAVER 3.0 software (http: / / www.caver.cz / ) was used to identify and analyze enzyme reaction channels, determining key amino acid sites near the channel entrance (positions 331 and 365). Primers (329-331-F, 361-365-R) were designed based on these key amino acid sites. The gene fragment amplified using these primers was then subjected to PCR amplification again, ultimately obtaining a mutant library with simultaneous mutations at all four sites. The specific steps are as follows:

[0073] Using the recombinant plasmid pET-32a-mal as a template, primers were designed based on the key amino acid sites mentioned above. The primers used are shown below:

[0074] 329-331-F: 5'-TGTCACATGTGDTATCNDTACCCGG-3';

[0075] 361-365-R: 5'-GGCACTGACAHNAGTTTCATTAHNGGTGCCG-3';

[0076] NDT represents any one of the twelve amino acids (R / N / D / C / G / H / I / L / F / S / Y / V). A PCR amplification program was designed using primers 329-331-F and 361-365-R: 95℃ denaturation for 3 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 10 min, 30 cycles; total extension at 72℃ for 30 s, yielding one gene fragment. Two large primers were used with the complementary strand of the gene fragment, with the following sequences:

[0077] Large primer F:

[0078] 5'-TGTCACATGGTGNDTATCNDTACCCCGGATCTGGGCAGTATTCACAACATCGTCGATGCGGTTCTTTACTGCAACAGCCACAGCATGGAAGCGTACCAGGGCGGCACCNDTAATGAAACTNDTGTCAGTGCC-3';

[0079] Large primer R:

[0080] 5'-GGCACTGACAHNAGTTTCATTAHNGGTGCCGCCCTGGTACGCTTCCATGCTGTGGCTGTTGCAGTAAAGAACCGCATCGACGATGTTGTGAATACTGCCCAGATCCGGGGTAHNGATAHNCACCATGTGACA-3'.

[0081] Using recombinant plasmid pET-32a-mal as a template, a large primer PCR amplification program was designed: 95℃ denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 7 min, 30 cycles; 72℃ total extension for 10 min. The full plasmid PCR amplification product was obtained.

[0082] After verification by 1% agarose gel electrophoresis, the cells were digested with the restriction enzyme DpnI. The digested product was then transformed into E. coli BL21(DE3) competent cells by heat shock to obtain a mutant library with simultaneous mutations at four sites.

[0083] (2) High-throughput screening of mutant libraries

[0084] The methylaspartate lyase (EcMAL) produced by recombinant bacteria containing EcMAL catalyzes the amination addition reaction of fumarate to generate aspartic acid. Utilizing the highly specific oxidation of D-aspartate by D-ASPO to iminosuccinic acid and the byproduct H₂O₂, and the property that horseradish peroxidase (HRP) can oxidize the chromogenic reagent 3',3-diaminobenzidine (DAB) to a reddish-brown compound in the presence of H₂O₂, a high-throughput solid-state colorimetric screening method was developed. The steps are as follows:

[0085] First, the mutants from the mutant library were plated onto ampicillin-resistant LB solid medium (the nylon membrane was attached to the surface of the LB solid medium, and the working concentration of ampicillin was 100 μg / mL) with a nylon membrane (purchased from GE Healthcare, USA) attached to it, and incubated overnight at 37°C and 180 rpm for 16 h. The nylon membrane with mutants was then transferred to LB solid medium containing the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) and ampicillin-resistant medium (working concentration of ampicillin 100 μg / mL, IPTG concentration 0.1 mg / mL), and induced at 30°C for 4 h to obtain the induced nylon membrane with mutants. This nylon membrane was then transferred to an empty culture dish and repeatedly frozen and thawed four times with liquid nitrogen to obtain the freeze-thawed nylon membrane with mutants. A piece of filter paper of the same size as the nylon membrane was then immersed in a pre-prepared mixture (500 mM Tris-HCl, 20 mM MgCl2, 500 mM... The mixture was prepared by adding NH4Cl, 10mM fumaric acid, 0.1mg / mL HRP, 0.1mM DAB, and 0.5mg / mL D-ASPO crude enzyme solution. The nylon membrane containing the mutant was then placed on filter paper after freeze-thaw cycles and incubated at 30℃ for at least 30 minutes. Single colonies showing a reddish-brown compound were the desired active mutants (i.e., mutant EcMAL-M1(Q329D / K331G / C361H / D365D), mutant EcMAL-M2(Q329R / K331I / C361R / D365L), mutant EcMAL-M3(Q329H / K331S / C361C / D365S), and mutant EcMAL-M4(Q329V / K331C / C361C / D365L)).

[0086] The D-ASPO gene has been disclosed in the article T Shouji, T Toshiyuki, K Yoshio et al. Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from theireast Cryptococcus humicola and characterization of the recombinant enzyme. DOI: 10.3923 / ajps.2007.399.402, and its gene sequence is shown in SEQ ID NO.11. The gene was synthesized at General Biosystems (Anhui) Co., Ltd., and introduced into E. coli using PET-32a(+) as a vector. BL21(DE3) competent cells were cultured in LB liquid medium containing ampicillin resistance (working concentration of ampicillin 100 μg / mL) at 37℃ and 180 rpm for 12 h. Then, 1% of the cells were transferred to fresh LB liquid medium containing ampicillin resistance and cultured at 37℃ and 180 rpm. When the cell concentration (OD600) reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM. The cells were cultured at 25℃ and 180 rpm for 8 h. The cells were then centrifuged at 4℃ and 8000 rpm for 5 min to obtain a precipitate. The precipitate was resuspended in Tris-HCl buffer (pH = 8.5, 500 mM) at a mass ratio of 1:10 (g:g), sonicated for 15 min, and centrifuged again at 4℃ and 10000 rpm for 10 min to obtain the supernatant, which is the crude D-ASPO enzyme solution.

[0087] 0.5 mg / mL D-ASPO crude enzyme solution means that 1 mL of the mixture contains 0.5 mg of D-ASPO crude enzyme solution.

[0088] (3) Validation of active mutants

[0089] Active mutants (i.e., mutants EcMAL-M1, EcMAL-M2, EcMAL-M3, and EcMAL-M4) were inoculated into LB liquid medium containing ampicillin resistance (working concentration of ampicillin 100 μg / mL; the same below), and cultured at 37℃ and 180 rpm for 12 h. Then, 1% of the inoculum was transferred to fresh LB liquid medium containing ampicillin resistance and cultured at 37℃ and 180 rpm. When the bacterial cell concentration (OD600) reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the culture was carried out at 16℃ and 180 rpm for 20 h. The culture was then centrifuged at 4℃ and 8000 rpm for 5 min to obtain a precipitate. The precipitate was then collected and combined with Tr... Resuspend the protein in Tris-HCl buffer (pH = 8.5, 100 mM) at a mass ratio of 1:10 (g:g), sonicate for 15 min, and centrifuge again at 4℃, 10000 rpm for 10 min to obtain the supernatant. Purify the target protein in the supernatant using a protein purification system (loading buffer: a mixture of Tris-HCl buffer and imidazole; wherein the pH of Tris-HCl buffer is 8.5 and the concentration is 100 mM; the concentration of imidazole is 20 mM. Elution buffer: a mixture of Tris-HCl buffer and imidazole; wherein the pH of Tris-HCl buffer is 8.5 and the concentration is 100 mM; the concentration of imidazole is 250 mM). The pure mutant enzyme EcMAL was finally obtained.

[0090] The purified mutant enzyme EcMAL was added to 5 mL of reaction solution at a final concentration of 0.5 mg / mL (the reaction solution consisted of 500 mM Tris-HCl, 20 mM MgCl2, 500 mM NH4Cl, 10 mM fumaric acid, pH 8.5). The reaction was carried out at 30 °C and 200 rpm for 20 h, terminated by boiling in a water bath for 5 min, and centrifuged at 12000 rpm for 10 min. The D-aspartic acid concentration was then determined by high-performance liquid chromatography (HPLC). The optimal mutant was determined based on the amount of D-aspartic acid produced. The above experiment was performed using recombinant E. coli BL21(DE3) / pET-32a-mal(EcMAL) as a control.

[0091] High performance liquid chromatography (HPLC): A Chiral AAOA 5u 150*4.6mm column was used. The mobile phase was a mixture of CuSO4 solution and isopropanol at a volume ratio of 95:5 (mL:mL), wherein the concentration of CuSO4 solution was 2mM and the isopropanol was chromatographic grade isopropanol. The flow rate was 1mL / min, the column temperature was 30℃, the injection volume was 20μL, and the peak area of ​​D-aspartic acid was determined at a wavelength of 254nm.

[0092] Table 2:

[0093]

[0094] The liquid chromatography-liquid phase diagram of L-aspartic acid and D-aspartic acid is shown below. Figure 1 As shown. The EcMAL produced by the methylaspartate lyase EcMAL mutants (mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3, and mutant EcMAL-M4) and the recombinant strain E. coli BL21(DE3) / pET-32a-mal(EcMAL) after the catalytic reaction using fumaric acid as a substrate yields L-aspartic acid and D-aspartic acid, respectively. Figure 2-6 As shown. From Figure 2 It can be seen that the EcMAL expressed by the recombinant strain E.coliBL21(DE3) / pET-32a-mal failed to produce D-aspartic acid in the fumarate reaction, instead producing only L-aspartic acid (conversion rate 85%, i.e., 10 mM fumarate ultimately produces 8.5 mM L-aspartic acid). This indicates that the methylaspartate lyase (EcMAL) catalyzes the amination addition reaction of fumarate to produce L-aspartic acid. Figure 3-6 It can be seen that the mutant enzyme EcMAL expressed by mutants EcMAL-M1, EcMAL-M2, EcMAL-M3, and EcMAL-M4 all produced D-aspartic acid in the reaction catalyzed by fumarate (among which, mutant EcMAL-M1...). Figure 3 Using 10 mM fumaric acid as a substrate, the conversion of fumaric acid to L / D-aspartic acid was 60% in yield, with the final concentrations of L-aspartic acid being 3.5 mM and D-aspartic acid being 2.5 mM. Therefore, the relative yields of L-aspartic acid were 58.3% and D-aspartic acid were 41.7%. The mutant EcMAL-M2 ( Figure 4 Using 10 mM fumaric acid as a substrate, the yield of fumaric acid to L / D-aspartic acid was 58%, with a final concentration of 3.8 mM for L-aspartic acid and 2.0 mM for D-aspartic acid, resulting in a relative yield of 65.5% for L-aspartic acid and 34.5% for D-aspartic acid; the mutant EcMAL-M3 ( Figure 5 Using 10 mM fumaric acid as a substrate, the yield of fumaric acid to L / D-aspartic acid was 57%, with a final concentration of 4.0 mM for L-aspartic acid and 1.7 mM for D-aspartic acid, resulting in a relative yield of 70.2% for L-aspartic acid and 29.8% for D-aspartic acid; the mutant EcMAL-M4 ( Figure 6(Using 10 mM fumaric acid as a substrate, the yield of fumaric acid to L / D-aspartic acid was 38%, and the final concentration of L-aspartic acid was 3.3 mM and the concentration of D-aspartic acid was 0.5 mM. Thus, the relative yield of L-aspartic acid was 86.8% and the relative yield of D-aspartic acid was 13.2% (see Table 2).

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. sequence list <110> South China University of Technology <120> A methylaspartate lyase EcMAL mutant, its preparation method and application <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 413 <212> PRT <213> Artificial Sequence <223> Amino acid sequence of methylaspartate lyase <400> 1 Met Lys Ile Lys Gln Ala Leu Phe Thr Ala Gly Tyr Ser Ser Phe Tyr 1 5 10 15 Phe Asp Asp Gln Gln Ala Ile Lys Asn Gly Ala Gly His Asp Gly Phe 20 25 30 Phe Tyr Thr Gly Glu Pro Val Thr Gln Gly Phe Asn Ala Val Arg Gln 35 40 45 Ala Gly Glu Cys Val Ser Val Gln Leu Ile Leu Glu Asn Gly Ala Val 50 55 60 Ala Val Gly Asp Cys Thr Ala Val Gln Tyr Ser Gly Ala Gly Gly Arg 65 70 75 80 Asp Pro Leu Phe Leu Ala Glu His Phe Ile Pro Phe Leu Asn Asp His 85 90 95 Ile Lys Pro Leu Leu Val Gly Arg Asp Val Asp Ala Phe Leu Pro Asn 100 105 110 Ala Arg Phe Phe Asp Lys Leu Arg Ile Asp Gly Asn Leu Leu His Thr 115 120 125 Ala Val Arg Tyr Gly Leu Ser Gln Ala Leu Leu Asp Ala Thr Ala Leu 130 135 140 Ala Thr Gly Arg Leu Lys Thr Glu Val Val Cys Asp Glu Trp Gln Leu 145 150 155 160 Pro Arg Val Ala Glu Ser Ile Pro Leu Phe Gly Gln Ser Gly Asp Asp 165 170 175 Arg Tyr Ile Ala Val Asp Lys Met Ile Leu Lys Gly Ile Asp Val Leu 180 185 190 Pro His Ala Leu Ile Asn Asn Val Glu Glu Lys Leu Gly Phe Lys Gly 195 200 205 Glu Lys Leu Arg Glu Tyr Val Arg Trp Leu Ser Asp Arg Ile Leu Ser 210 215 220 Lys Arg Thr Ser Ala Arg Tyr His Pro Thr Leu His Ile Asp Val Tyr 225 230 235 240 Gly Thr Ile Gly Leu Ile Phe Asp Met Asp Pro Leu Arg Cys Ala Gln 245 250 255 Tyr Ile Ala Ser Leu Glu Lys Glu Ala Gln Gly Leu Pro Leu Tyr Ile 260 265 270 Glu Gly Pro Val Asp Ala Gly Asn Lys Pro Asp Gln Ile Arg Leu Leu 275 280 285 Thr Ala Ile Thr Lys Glu Leu Thr Arg Leu Gly Ser Gly Val Lys Ile 290 295 300 Val Ala Asp Glu Trp Cys Asn Thr Tyr Gln Asp Ile Val Asp Phe Thr 305 310 315 320 Asp Ala Ala Ser Cys His Met Val Gln Ile Lys Thr Pro Asp Leu Gly 325 330 335 Ser Ile His Asn Ile Val Asp Ala Val Leu Tyr Cys Asn Ser His Ser 340 345 350 Met Glu Ala Tyr Gln Gly Gly Thr Cys Asn Glu Thr Asp Val Ser Ala 355 360 365 Arg Thr Cys Val His Val Ala Leu Ala Ala Arg Pro Met Arg Met Leu 370 375 380 Val Lys Pro Gly Met Gly Phe Asp Glu Gly Leu Asp Ile Val Phe Asn 385 390 395 400 Glu Met Asn Arg Thr Ile Ala Leu Leu Gln Ala Lys Asp 405 410 <210> 2 <211> 1242 <212> DNA / RNA <213> Artificial Sequence <223> Nucleotide sequence of the EcMAL enzyme gene <400> 2 atgaaaataa aacaggctct gttcaccgct ggctactcct cattctattt cgatgaccag 60 caggcgataa aaaacggagc gggtcatgac ggcttttttt ataccgggga gccagtaaca 120 caggggttta acgccgtacg tcaggccggg gagtgcgtat cggtacagtt gattctggaa 180 aacggcgcgg tcgccgtagg tgactgtact gccgtacagt attccggggc aggcggtcgc 240 gatccactgt tcctcgcaga gcactttatt ccgttcctca acgaccatat caagccatta 300 ctggtaggcc gcgatgtgga tgctttcctg ccgaatgccc gtttcttcga caaattgcgt 360 attgacggca acttgctgca taccgccgtg cgctacggat tatcacaggc gctgcttgat 420 gctaccgcgc tggcaaccgg ccgtctgaaa actgaagtgg tctgtgatga atggcagttg 480 ccacgcgtgg cggaatccat tccattattt ggtcagagcg gcgacgatcg atatatcgcc 540 gtcgataaga tgatccttaa father gtgctgcccc atgcgctgat fathercgtc gaagagaagc tgggctttaa aggtgaaaaa ctgcgcgaat atgtccgctg gttgtcggat cgcattctaa gcaagcgcac cagcgcacgc taccacccta ccctgcacat cgatgtatac ggcactatcg gtctgatctt cgatatggat ccgcttcgct gtgcgcaata catcgccagc 780 ctggaaaaag aagcgcaagg cctgccgctc tacatcgaag ggccggtcga tgccggtaac aagcccgatc aaattcgcct gctgaccgcg attackaag agctgacgcg cctcggttcc 900 ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960 1020. gatgctgcca gttgtcacat ggtgcaaatc aaaaccccgg atctgggcag tattcacaac atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080. tgcaatgaaa ctgatgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140 atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac 1200 gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa 1242 <210> 3 <211> 413 <212> PRT <213> Artificial Sequence <223> Amino acid sequence of mutant EcMAL-M1 <400> 3 Met Lys Ile Lys Gln Ala Leu Phe Thr Ala Gly Tyr Ser Ser Phe Tyr 1 5 10 15 Phe Asp Asp Gln Gln Ala Ile Lys Asn Gly Ala Gly His Asp Gly Phe 20 25 30 Phe Tyr Thr Gly Glu Pro Val Thr Gln Gly Phe Asn Ala Val Arg Gln 35 40 45 Ala Gly Glu Cys Val Ser Val Gln Leu Ile Leu Glu Asn Gly Ala Val 50 55 60 Ala Val Gly Asp Cys Thr Ala Val Gln Tyr Ser Gly Ala Gly Gly Arg 65 70 75 80 Asp Pro Leu Phe Leu Ala Glu His Phe Ile Pro Phe Leu Asn Asp His 85 90 95 Ile Lys Pro Leu Leu Val Gly Arg Asp Val Asp Ala Phe Leu Pro Asn 100 105 110 Ala Arg Phe Phe Asp Lys Leu Arg Ile Asp Gly Asn Leu Leu His Thr 115 120 125 Ala Val Arg Tyr Gly Leu Ser Gln Ala Leu Leu Asp Ala Thr Ala Leu 130 135 140 Ala Thr Gly Arg Leu Lys Thr Glu Val Val Cys Asp Glu Trp Gln Leu 145 150 155 160 Pro Arg Val Ala Glu Ser Ile Pro Leu Phe Gly Gln Ser Gly Asp Asp 165 170 175 Arg Tyr Ile Ala Val Asp Lys Met Ile Leu Lys Gly Ile Asp Val Leu 180 185 190 Pro His Ala Leu Ile Asn Asn Val Glu Glu Lys Leu Gly Phe Lys Gly 195 200 205 Glu Lys Leu Arg Glu Tyr Val Arg Trp Leu Ser Asp Arg Ile Leu Ser 210 215 220 Lys Arg Thr Ser Ala Arg Tyr His Pro Thr Leu His Ile Asp Val Tyr 225 230 235 240 Gly Thr Ile Gly Leu Ile Phe Asp Met Asp Pro Leu Arg Cys Ala Gln 245 250 255 Tyr Ile Ala Ser Leu Glu Lys Glu Ala Gln Gly Leu Pro Leu Tyr Ile 260 265 270 Glu Gly Pro Val Asp Ala Gly Asn Lys Pro Asp Gln Ile Arg Leu Leu 275 280 285 Thr Ala Ile Thr Lys Glu Leu Thr Arg Leu Gly Ser Gly Val Lys Ile 290 295 300 Val Ala Asp Glu Trp Cys Asn Thr Tyr Gln Asp Ile Val Asp Phe Thr 305 310 315 320 Asp Ala Ala Ser Cys His Met Val Asp Ile Gly Thr Pro Asp Leu Gly 325 330 335 Ser Ile His Asn Ile Val Asp Ala Val Leu Tyr Cys Asn Ser His Ser 340 345 350 Met Glu Ala Tyr Gln Gly Gly Thr His Asn Glu Thr Asp Val Ser Ala 355 360 365 Arg Thr Cys Val His Val Ala Leu Ala Ala Arg Pro Met Arg Met Leu 370 375 380 Val Lys Pro Gly Met Gly Phe Asp Glu Gly Leu Asp Ile Val Phe Asn 385 390 395 400 Glu Met Asn Arg Thr Ile Ala Leu Leu Gln Ala Lys Asp 405 410 <210> 4 <211> 1242 <212> DNA / RNA <213> Artificial Sequence <223> Nucleotide sequence of mutant EcMAL-M1 gene <400> 4 atgaaaataa aacaggctct gttcaccgct ggctactcct cattctattt cgatgaccag 60 caggcgataa aaaacggagc gggtcatgac ggcttttttt ataccgggga gccagtaaca 120 caggggttta acgccgtacg tcaggccggg gagtgcgtat cggtacagtt gattctggaa 180 aacggcgcgg tcgccgtagg tgactgtact gccgtacagt attccggggc aggcggtcgc 240 gatccactgt tcctcgcaga gcactttatt ccgttcctca acgaccatat caagccatta 300 ctggtaggcc gcgatgtgga tgctttcctg ccgaatgccc gtttcttcga caaattgcgt 360 attgacggca acttgctgca taccgccgtg cgctacggat tatcacaggc gctgcttgat 420 gctaccgcgc tggcaaccgg ccgtctgaaa actgaagtgg tctgtgatga atggcagttg 480 ccacgcgtgg cggaatccat tccattattt ggtcagagcg gcgacgatcg atatatcgcc 540 gtcgataaga tgatccttaa aggcatcgac gtgctgcccc atgcgctgat taataacgtc 600 gaagagaagc tgggctttaa aggtgaaaaa ctgcgcgaat atgtccgctg gttgtcggat 660 cgcattctaa gcaagcgcac cagcgcacgc taccacccta ccctgcacat cgatgtatac ggcactatcg gtctgatctt cgatatggat ccgcttcgct gtgcgcaata catcgccagc 780 ctggaaaaag aagcgcaagg cctgccgctc tacatcgaag ggccggtcga tgccggtaac aagcccgatc aaattcgcct gctgaccgcg attackaag agctgacgcg cctcggttcc 900 ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960 1020. gatgctgcca gttgtcacat ggtggatatc ggtaccccgg atctgggcag tattcacaac atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080. cataatgaaa ctgatgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140 atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa <210> 5 <211> 413 <212> PRT <213> Artificial Sequence <223> The EcMAL‐M2 carrier has a smooth surface <400> 5 Met Lys Ile Lys Gln Ala Leu Phe Thr Ala Gly Tyr Ser Ser Phe Tyr 1 5 10 15 Phe Asp Asp Gln Gln Ala Ile Lys Asn Gly Ala Gly His Asp Gly Phe 20 25 30 Phe Tyr Thr Gly Glu Pro Val Thr Gln Gly Phe Asn Ala Val Arg Gln 35 40 45 Ala Gly Glu Cys Val Ser Val Gln Leu Ile Leu Glu Asn Gly Ala Val 50 55 60 Ala Val Gly Asp Cys Thr Ala Val Gln Tyr Ser Gly Ala Gly Gly Arg 65 70 75 80 Asp Pro Leu Phe Leu Ala Glu His Phe Ile Pro Phe Leu Asn Asp His 85 90 95 Ile Lys Pro Leu Leu Val Gly Arg Asp Val Asp Ala Phe Leu Pro Asn 100 105 110 Ala Arg Phe Phe Asp Lys Leu Arg Ile Asp Gly Asn Leu Leu His Thr 115 120 125 Ala Val Arg Tyr Gly Leu Ser Gln Ala Leu Leu Asp Ala Thr Ala Leu 130 135 140 Ala Thr Gly Arg Leu Lys Thr Glu Val Val Cys Asp Glu Trp Gln Leu 145 150 155 160 Pro Arg Val Ala Glu Ser Ile Pro Leu Phe Gly Gln Ser Gly Asp Asp 165 170 175 Arg Tyr Ile Ala Val Asp Lys Met Ile Leu Lys Gly Ile Asp Val Leu 180 185 190 Pro His Ala Leu Ile Asn Asn Val Glu Glu Lys Leu Gly Phe Lys Gly 195 200 205 Glu Lys Leu Arg Glu Tyr Val Arg Trp Leu Ser Asp Arg Ile Leu Ser 210 215 220 Lys Arg Thr Ser Ala Arg Tyr His Pro Thr Leu His Ile Asp Val Tyr 225 230 235 240 Gly Thr Ile Gly Leu Ile Phe Asp Met Asp Pro Leu Arg Cys Ala Gln 245 250 255 Tyr Ile Ala Ser Leu Glu Lys Glu Ala Gln Gly Leu Pro Leu Tyr Ile 260 265 270 Glu Gly Pro Val Asp Ala Gly Asn Lys Pro Asp Gln Ile Arg Leu Leu 275 280 285 Thr Ala Ile Thr Lys Glu Leu Thr Arg Leu Gly Ser Gly Val Lys Ile 290 295 300 Val Ala Asp Glu Trp Cys Asn Thr Tyr Gln Asp Ile Val Asp Phe Thr 305 310 315 320 Asp Ala Ala Ser Cys His Met Val Arg Ile Ile Thr Pro Asp Leu Gly 325 330 335 Ser Ile His Asn Ile Val Asp Ala Val Leu Tyr Cys Asn Ser His Ser 340 345 350 Met Glu Ala Tyr Gln Gly Gly Thr Arg Asn Glu Thr Leu Val Ser Ala 355 360 365 Arg Thr Cys Val His Val Ala Leu Ala Ala Arg Pro Met Arg Met Leu 370 375 380 Val Lys Pro Gly Met Gly Phe Asp Glu Gly Leu Asp Ile Val Phe Asn 385 390 395 400 Glu Met Asn Arg Thr Ile Ala Leu Leu Gln Ala Lys Asp 405 410 <210> 6 <211> 1242 <212> DNA / RNA <213> Artificial Sequence <223> Nucleotide sequence of the mutant EcMAL-M2 gene <400> 6 atgaaaataa aacaggctct gttcaccgct ggctactcct cattctattt cgatgaccag 60 caggcgataa aaaacggagc gggtcatgac ggcttttttt ataccgggga gccagtaaca 120 caggggttta acgccgtacg tcaggccggg gagtgcgtat cggtacagtt gattctggaa 180 aacggcgcgg tcgccgtagg tgactgtact gccgtacagt attccggggc aggcggtcgc 240 gatccactgt tcctcgcaga gcactttatt ccgttcctca acgaccatat caagccatta 300 ctggtaggcc gcgatgtgga tgctttcctg ccgaatgccc gtttcttcga caaattgcgt 360 attgacggca acttgctgca taccgccgtg cgctacggat tatcacaggc gctgcttgat 420 gctaccgcgc tggcaaccgg ccgtctgaaa actgaagtgg tctgtgatga atggcagttg 480 ccacgcgtgg cggaatccat tccattattt ggtcagagcg gcgacgatcg atatatcgcc 540 gtcgataaga tgatccttaa aggcatcgac gtgctgcccc atgcgctgat taataacgtc 600 gaagagaagc tgggctttaa aggtgaaaaa ctgcgcgaat atgtccgctg gttgtcggat 660 cgcattctaa gcaagcgcac cagcgcacgc taccacccta ccctgcacat cgatgtatac 720 ggcactatcg gtctgatctt cgatatggat ccgcttcgct gtgcgcaata catcgccagc 780 ctggaaaaag aagcgcaagg cctgccgctc tacatcgaag ggccggtcga tgccggtaac 840 aagcccgatc aaattcgcct gctgaccgcg attackaag agctgacgcg cctcggttcc 900 ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960 1020. gatgctgcca gttgtcacat ggtgcgtatc ataaccccgg atctgggcag tattcacaac atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080. cgtaatgaaa ctcttgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140 atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa <210> 7 <211> 413 <212> PRT <213> Artificial Sequence <223> The EcMAL‐M3 carrier has a smooth surface <400> 7 Met Lys Ile Lys Gln Free Mp3 Download 1 5 10 15 Phe Asp Asp Gln Gln Ala Ile Lys Asn Gly Ala Gly His Asp Gly Phe 20 25 30 Phe Tyr Thr Gly Glu Pro Val Thr Gln Gly Phe Asn Ala Val Arg Gln 35 40 45 Ala Gly Glu Cys Val Ser Val Gln Leu Ile Leu Glu Asn Gly Ala Val 50 55 60 Ala Val Gly Asp Cys Thr Ala Val Gln Tyr Ser Gly Ala Gly Gly Arg 65 70 75 80 Asp Pro Leu Phe Leu Ala Glu His Phe Ile Pro Phe Leu Asn Asp His 85 90 95 Ile Lys Pro Leu Leu Val Gly Arg Asp Val Asp Ala Phe Leu Pro Asn 100 105 110 Ala Arg Phe Phe Asp Lys Leu Arg Ile Asp Gly Asn Leu Leu His Thr 115 120 125 Ala Val Arg Tyr Gly Leu Ser Gln Ala Leu Leu Asp Ala Thr Ala Leu 130 135 140 Ala Thr Gly Arg Leu Lys Thr Glu Val Val Cys Asp Glu Trp Gln Leu 145 150 155 160 Pro Arg Val Ala Glu Ser Ile Pro Leu Phe Gly Gln Ser Gly Asp Asp 165 170 175 Arg Tyr Ile Ala Val Asp Lys Met Ile Leu Lys Gly Ile Asp Val Leu 180 185 190 Pro His Ala Leu Ile Asn Asn Val Glu Glu Lys Leu Gly Phe Lys Gly 195 200 205 Glu Lys Leu Arg Glu Tyr Val Arg Trp Leu Ser Asp Arg Ile Leu Ser 210 215 220 Lys Arg Thr Ser Ala Arg Tyr His Pro Thr Leu His Ile Asp Val Tyr 225 230 235 240 Gly Thr Ile Gly Leu Ile Phe Asp Met Asp Pro Leu Arg Cys Ala Gln 245 250 255 Tyr Ile Ala Ser Leu Glu Lys Glu Ala Gln Gly Leu Pro Leu Tyr Ile 260 265 270 Glu Gly Pro Val Asp Ala Gly Asn Lys Pro Asp Gln Ile Arg Leu Leu 275 280 285 Thr Ala Ile Thr Lys Glu Leu Thr Arg Leu Gly Ser Gly Val Lys Ile 290 295 300 Val Ala Asp Glu Trp Cys Asn Thr Tyr Gln Asp Ile Val Asp Phe Thr 305 310 315 320 Asp Ala Ala Ser Cys His Met Val His Ile Ser Thr Pro Asp Leu Gly 325 330 335 Ser Ile His Asn Ile Val Asp Ala Val Leu Tyr Cys Asn Ser His Ser 340 345 350 Met Glu Ala Tyr Gln Gly Gly Thr Cys Asn Glu Thr Ser Val Ser Ala 355 360 365 Arg Thr Cys Val His Val Ala Leu Ala Ala Arg Pro Met Arg Met Leu 370 375 380 Val Lys Pro Gly Met Gly Phe Asp Glu Gly Leu Asp Ile Val Phe Asn 385 390 395 400 Glu Met Asn Arg Thr Ile Ala Leu Leu Gln Ala Lys Asp 405 410 <210> 8 <211> 1242 <212> DNA / RNA <213> Artificial Sequence <223> Nucleotide sequence of mutant EcMAL-M3 gene <400> 8 atgaaaataa aacaggctct gttcaccgct ggctactcct cattctattt cgatgaccag 60 caggcgataa aaaacggagc gggtcatgac ggcttttttt ataccgggga gccagtaaca 120 caggggttta acgccgtacg tcaggccggg gagtgcgtat cggtacagtt gattctggaa 180 aacggcgcgg tcgccgtagg tgactgtact gccgtacagt attccggggc aggcggtcgc 240 gatccactgt tcctcgcaga gcactttatt ccgttcctca acgaccatat caagccatta 300 ctggtaggcc gcgatgtgga tgctttcctg ccgaatgccc gtttcttcga caaattgcgt 360 attgacggca acttgctgca taccgccgtg cgctacggat tatcacaggc gctgcttgat gctaccgcgc tggcaaccgg ccgtctgaaa actgaagtgg tctgtgatga atggcagttg 480 ccacgcgtgg cggaatccat tccattattt ggtcagagcg gcgacgatcg atatatcgcc 540 gtcgataaga tgatccttaa father gtgctgcccc atgcgctgat fathercgtc gaagagaagc tgggctttaa aggtgaaaaa ctgcgcgaat atgtccgctg gttgtcggat cgcattctaa gcaagcgcac cagcgcacgc taccacccta ccctgcacat cgatgtatac ggcactatcg gtctgatctt cgatatggat ccgcttcgct gtgcgcaata catcgccagc 780 ctggaaaaag aagcgcaagg cctgccgctc tacatcgaag ggccggtcga tgccggtaac aagcccgatc aaattcgcct gctgaccgcg attackaag agctgacgcg cctcggttcc 900 ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960 gatgctgcca gttgtcacat ggtgcatatc agtaccccgg atctgggcag tattcacaac atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080. tgtaatgaaa ctagtgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140 atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac 1200 gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa 1242 <210> 9 <211> 413 <212> PRT <213> Artificial Sequence <223> Amino acid sequence of mutant EcMAL-M4 <400> 9 Met Lys Ile Lys Gln Ala Leu Phe Thr Ala Gly Tyr Ser Ser Phe Tyr 1 5 10 15 Phe Asp Asp Gln Gln Ala Ile Lys Asn Gly Ala Gly His Asp Gly Phe 20 25 30 Phe Tyr Thr Gly Glu Pro Val Thr Gln Gly Phe Asn Ala Val Arg Gln 35 40 45 Ala Gly Glu Cys Val Ser Val Gln Leu Ile Leu Glu Asn Gly Ala Val 50 55 60 Ala Val Gly Asp Cys Thr Ala Val Gln Tyr Ser Gly Ala Gly Gly Arg 65 70 75 80 Asp Pro Leu Phe Leu Ala Glu His Phe Ile Pro Phe Leu Asn Asp His 85 90 95 Ile Lys Pro Leu Leu Val Gly Arg Asp Val Asp Ala Phe Leu Pro Asn 100 105 110 Ala Arg Phe Phe Asp Lys Leu Arg Ile Asp Gly Asn Leu Leu His Thr 115 120 125 Ala Val Arg Tyr Gly Leu Ser Gln Ala Leu Leu Asp Ala Thr Ala Leu 130 135 140 Ala Thr Gly Arg Leu Lys Thr Glu Val Val Cys Asp Glu Trp Gln Leu 145 150 155 160 Pro Arg Val Ala Glu Ser Ile Pro Leu Phe Gly Gln Ser Gly Asp Asp 165 170 175 Arg Tyr Ile Ala Val Asp Lys Met Ile Leu Lys Gly Ile Asp Val Leu 180 185 190 Pro His Ala Leu Ile Asn Asn Val Glu Glu Lys Leu Gly Phe Lys Gly 195 200 205 Glu Lys Leu Arg Glu Tyr Val Arg Trp Leu Ser Asp Arg Ile Leu Ser 210 215 220 Lys Arg Thr Ser Ala Arg Tyr His Pro Thr Leu His Ile Asp Val Tyr 225 230 235 240 Gly Thr Ile Gly Leu Ile Phe Asp Met Asp Pro Leu Arg Cys Ala Gln 245 250 255 Tyr Ile Ala Ser Leu Glu Lys Glu Ala Gln Gly Leu Pro Leu Tyr Ile 260 265 270 Glu Gly Pro Val Asp Ala Gly Asn Lys Pro Asp Gln Ile Arg Leu Leu 275 280 285 Thr Ala Ile Thr Lys Glu Leu Thr Arg Leu Gly Ser Gly Val Lys Ile 290 295 300 Val Ala Asp Glu Trp Cys Asn Thr Tyr Gln Asp Ile Val Asp Phe Thr 305 310 315 320 Asp Ala Ala Ser Cys His Met Val Val Ile Cys Thr Pro Asp Leu Gly 325 330 335 Ser Ile His Asn Ile Val Asp Ala Val Leu Tyr Cys Asn Ser His Ser 340 345 350 Met Glu Ala Tyr Gln Gly Gly Thr Cys Asn Glu Thr Leu Val Ser Ala 355 360 365 Arg Thr Cys Val His Val Ala Leu Ala Ala Arg Pro Met Arg Met Leu 370 375 380 Val Lys Pro Gly Met Gly Phe Asp Glu Gly Leu Asp Ile Val Phe Asn 385 390 395 400 Glu Met Asn Arg Thr Ile Ala Leu Leu Gln Ala Lys Asp 405 410 <210> 10 <211> 1242 <212> DNA / RNA <213> Artificial Sequence <223> Nucleotide sequence of mutant EcMAL-M4 gene <400> 10 atgaaaataa aacaggctct gttcaccgct ggctactcct cattctattt cgatgaccag 60 caggcgataa aaaacggagc gggtcatgac ggcttttttt ataccgggga gccagtaaca 120 caggggttta acgccgtacg tcaggccggg gagtgcgtat cggtacagtt gattctggaa 180 aacggcgcgg tcgccgtagg tgactgtact gccgtacagt attccggggc aggcggtcgc 240 gatccactgt tcctcgcaga gcactttatt ccgttcctca acgaccatat caagccatta 300 ctggtaggcc gcgatgtgga tgctttcctg ccgaatgccc gtttcttcga caaattgcgt 360 attgacggca acttgctgca taccgccgtg cgctacggat tatcacaggc gctgcttgat 420 gctaccgcgc tggcaaccgg ccgtctgaaa actgaagtgg tctgtgatga atggcagttg 480 ccacgcgtgg cggaatccat tccattattt ggtcagagcg gcgacgatcg atatatcgcc 540 gtcgataaga tgatccttaa aggcatcgac gtgctgcccc atgcgctgat taataacgtc 600 gaagagaagc tgggctttaa aggtgaaaaa ctgcgcgaat atgtccgctg gttgtcggat cgcattctaa gcaagcgcac cagcgcacgc taccacccta ccctgcacat cgatgtatac ggcactatcg gtctgatctt cgatatggat ccgcttcgct gtgcgcaata catcgccagc 780 ctggaaaaag aagcgcaagg cctgccgctc tacatcgaag ggccggtcga tgccggtaac aagcccgatc aaattcgcct gctgaccgcg attackaag agctgacgcg cctcggttcc 900 ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960 1020. gatgctgcca gttgtcacat ggtggttatc tgtaccccgg atctgggcag tattcacaac atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080. tgtaatgaaa ctcttgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140 atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa <210> 11 <211> 1113 <212> DNA / RNA <213> Artificial Sequence Gene sequence of D-ASPO <400> 11 atgcccccct cggaccccat catcgtcctc ggcgcgggcg tgataggcct caccacggcc 60 gtgcggctgc tcgaggcgca ccttggcgcg aacgtccaca tactcgcgga tcactggcct 120 tcggacgcgc tggacgcgca gtacgccagt acgatcgccg gcgcacacca cctcagcttt 180 gcggatgacg gcgacgcgcg ccagcgccgc tgggacatgc ggacgtttga cgtgctgtac 240 gacgagtgga aggccgtcgg ggagaggacc gggcttatgg cgctcacgca gaccgagatg 300 tgggagggcg cgacgtcgca tctcgccgtg tacgagggga acccagattt ccgcgtgctc 360 gacccgcgta ccgccccgtg cagcaacatc acccacatgg tgtccttcac gagcctgacc 420 atcgcgccga cggtgtacct cgccgcgctg gaagcccgcg tgcgcgacct cggcgcgaag 480 ctgcaccgtg cccacgtccc atcgctgggc gcgctgcgca ccgacccggc cctgctggcg 540 ctgtacactc gccccccggc cgcggtgttc gtctgcgccg gcctcggcgc gcgccacctc 600 gtgcccgcgc ctgaggccgc cgcgctcttc cccacccgcg ggcaggtcgt cgtcgtccgc 660 gcgccgtgga tgcgcgcagg gttcacgcgc caggtcggct cgctcggcgg cggcgagggc 720 ggcacgcgca cgtacattat cccgcggtgt aacggcgagg tcgtgcttgg cggcacgatg 780 gagcagggcg actggacgcc gtacccccgc gacgagacag tcacggacat cctcacgcgc 840 gcgctgcaga tctgcccgga catcgcgcca ccgtacgcgc gctcctggcc caaggacgac 9OO caggtggccg cgctgcgttc cattgtcgtg cgcgatgcgg tcggctttag gcctagccgc 960 gccggcggcg cccgggtcgc gctcgcctcg gcggccggca tgcgcgtcgt gtataactat 1020 ggccatggcg gcgcggggtg gcaaagctgc tggggctgtg cagaggacgc ggtggcgctg 1080 tgggccgggg gggctggggg tgcacggctg tag 1113 <210> 12 <211> 47 <212> DNA / RNA <213> Artificial Sequence <223> mal‑F <400> 12 gctgatatcg gatccgaatt catgaaaata aaacaggctc tgttcac 47 <210> 13 <211> 44 <212> DNA / RNA <213> Artificial Sequence <223> mal‑R <400> 13 gtggtggtgg tggtgctcga gttaatcctt agcctgcaac agcg 44

Claims

1. A methylaspartate lyase EcMAL mutant, EcMAL-M1, characterized in that, The amino acid sequence of the mutant EcMAL-M1 is shown in SEQ ID NO:

3.

2. The EcMAL-M1 mutant of methylaspartate lyase according to claim 1, characterized in that, The nucleotide sequence encoding the mutant EcMAL-M1 gene is shown in SEQ ID NO:

4.

3. The EcMAL mutant of methylaspartate lyase EcMAL according to claim 1, characterized in that, The methylaspartate lyase of the mutant before mutation was derived from Escherichia coli O157:H7.

4. The method for preparing the EcMAL mutant EcMAL-M1 of methylaspartate lyase according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. The methylaspartate lyase gene was ligated into a plasmid to obtain a recombinant plasmid; S2. Design mutation primers, use the mutation primers and the recombinant plasmid as a template for PCR amplification, and then use the PCR amplification product as a large primer for full plasmid amplification to obtain the mutation product. Transform the mutation product into the host cell to obtain the mutant library. S3. High-throughput screening of the mutant library yielded the EcMAL mutant of methylaspartate lyase, EcMAL-M1.

5. The preparation method according to claim 4, characterized in that, In step S2, the mutation primers are 329-331-F and 361-365-R: 329-331-F: 5'-TGTCACATGTGDTATCNDTACCCGG-3'; 361-365-R: 5'-GGCACTGACAHNAGTTTCATTAHNGGTGCCG-3'; NDT represents any one of the twelve amino acids: R, N, D, C, G, H, I, L, F, S, Y, and V. In step S2, the large primer is: Large primer F: 5'-TGTCACATGGTGNDTATCNDTACCCCGGATCTGGGCAGTATTCACAACATCGTCGATGCGGTTCTTTACTGCAACAGCCACAGCATGGAAGCGTACCAGGGCGGCACCNDTAATGAAACTNDTGTCAGTGCC-3'; Large primer R: 5'-GGCACTGACAHNAGTTTCATTAHNGGTGCCGCCCTGGTACGCTTCCATGCTGTGGCTGTTGCAGTAAAGAACCGCATCGACGATGTTGTGAATACTGCCCAGATCCGGGGTAHNGATAHNCACCATGTGACA-3'.

6. The preparation method according to claim 4, characterized in that, In step S3, the method for high-throughput screening of the mutant library to obtain the EcMAL mutant of methylaspartate lyase is as follows: the mutant in the mutant library is coated on a culture medium with a nylon membrane attached and cultured and induced to obtain an induced nylon membrane with the mutant; the induced nylon membrane with the mutant is subjected to freeze-thaw treatment to obtain a freeze-thawed nylon membrane with the mutant; the freeze-thawed nylon membrane with the mutant is covered on filter paper containing a mixed solution and allowed to stand; the appearance of a reddish-brown single colony is the EcMAL mutant of methylaspartate lyase; wherein: the mixed solution is a mixture containing horseradish peroxidase and 3',3-diaminobenzidine; The mixture consists of the following components: 500mM Tris-HCl, 20mM MgCl2, 500mM NH4Cl, 10mM fumaric acid, 0.1mg / mL HRP, 0.1mM DAB and 0.5mg / mL D-ASPO crude enzyme solution; The gene sequence of D-ASPO is shown in SEQ ID NO.

11.

7. The preparation method according to claim 6, characterized in that, The crude D-ASPO enzyme solution was prepared by the following method: the D-ASPO gene was introduced into Escherichia coli for induced expression and isolation, thus obtaining the crude D-ASPO enzyme solution; The filter paper containing the mixture is obtained by immersing the filter paper in the mixture. The induction conditions are: induction at 25-35℃ for 3-8 hours; The conditions for static incubation are: static incubation at 20–40°C for at least 30 minutes.

8. The use of the EcMAL mutant EcMAL-M1 of the methylaspartate lyase according to any one of claims 1 to 3 in the preparation of D-aspartic acid.

9. A method for preparing D-aspartic acid using the EcMAL mutant EcMAL-M1 methylaspartate lyase according to any one of claims 1 to 3, characterized in that, Includes the following steps: The mutant methylaspartate lyase EcMAL was induced, cultured, isolated, and purified to obtain the mutant enzyme EcMAL. The mutant enzyme EcMAL was then added to the reaction solution to carry out the reaction, thus obtaining D-aspartic acid. The reaction solution consists of the following components: 500mM Tris-HCl, 20mM MgCl2, 500mM NH4Cl, 10mM fumaric acid, pH 8.5; The reaction conditions are: 20–40°C, 100–300 rpm for 10–30 h.

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