CN115896080A - Methyl aspartic acid lyase EcMAL mutant and preparation method and application thereof - Google Patents
Methyl aspartic acid lyase EcMAL mutant and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a methyl aspartate lyase EcMAL mutant and a preparation method and application thereof, belonging to the technical field of enzyme engineering. The methyl aspartate lyase EcMAL mutant is any one of a mutant EcMAL-M1, a mutant EcMAL-M2, a mutant EcMAL-M3 and a mutant EcMAL-M4. The invention obtains the methyl aspartate lyase EcMAL mutant by carrying out mutation on 329 th site, 331 th site, 361 th site and 365 th site of the methyl aspartate lyase and combining a high-throughput screening method. The mutant changes the current situation that the conventional recombinase only produces L-aspartic acid, makes the production of D-aspartic acid possible by using the methyl aspartic acid lyase as a biocatalyst, and is expected to be used in the pharmaceutical industry.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a methyl aspartic acid lyase EcMAL mutant and a preparation method and application thereof.
Background
Chirality is one of the essential attributes widely existing in nature, and chiral drugs have important applications in the field of drug synthesis and development. The various amino acids (other than glycine) that make up the human body also have enantiomeric forms L and D that are mirror images of each other. Natural L-amino acids constitute the basic skeleton of proteins, and D-amino acids have a unique structure and play an important role in the fields of medicines, foods, and the like. D-aspartic acid (D-Asp) is one of the D-amino acids, commonly used as precursor and intermediate in the synthesis of pharmaceuticals. The amino acid penicillin injection medicine aspoxicillin which is clinically used for the first time; d-aspartic acid-beta-hydroxylamine (DAH) and the like which are medicines synthesized by taking D-Asp as a precursor and used for treating virus infection and resisting tumors. In addition, D-amino acids are not easily degraded in vivo, and thus are not likely to develop corresponding drug resistance. This property makes it a great step in the synthesis of enzyme inhibitors, and substituting L-amino acids in polypeptide drugs will greatly prolong the half-life of polypeptide drugs and reduce side effects. In food, D-Asp is also used as a substitute for preservatives, taste modifiers and the like, and is widely used.
The technology for producing D-aspartic acid abroad, especially in Japan, is always at the forefront of the world, and is only rarely reported at home. The existing methods for preparing D-aspartic acid mainly comprise physical methods, chemical methods and biological methods. Wherein, the physical method is mainly to obtain corresponding pure products through crystallization and separation. The chemical method is divided into asymmetric synthesis and resolution. However, the physical method and the chemical method have the problems of complicated process, long steps, substandard yield and purity and the like, and are difficult to apply on a large scale. Biological methods are divided into biological resolution and asymmetric synthesis, and in the prior art, L-aspartate-alpha-decarboxylase, L-aspartate-beta-decarboxylase and aspartase are generally prepared by biological methods, however, the yield of D-aspartate is influenced because byproducts are generated in the biological resolution reaction. Asymmetric synthesis requires participation of multiple enzymes, and the yield is low at present, so that the method is lack of industrial application value. In the current research, natural methyl aspartate lyase is used for preparing L-aspartic acid, and no report of generating D-aspartic acid is found at present.
Disclosure of Invention
The primary objective of the present application is to provide a mutant of methyl aspartate lyase EcMAL.
Another objective of the present application is to provide a method for preparing the above-mentioned mutant EcMAL of methyl aspartate lyase.
Still another object of the present invention is to provide the use of the above-mentioned mutant EcMAL of methyl aspartate lyase.
The final object of the present application is to provide a method for preparing D-aspartic acid using the mutant EcMAL which is a methyl aspartate lyase.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a methyl aspartate lyase EcMAL mutant, which is any one of a mutant EcMAL-M1, a mutant EcMAL-M2, a mutant EcMAL-M3 and a mutant EcMAL-M4;
the mutant EcMAL-M1 is characterized in that the amino acid sequence of the mutant is methyl aspartic acid lyase shown as SEQ ID NO.1, wherein the 329 rd glutamine is mutated into aspartic acid, the 331 th lysine is mutated into glycine, the 361 th cysteine is mutated into histidine, and the 365 th aspartic acid is mutated into aspartic acid;
the mutant EcMAL-M2 is characterized in that the amino acid sequence of the mutant is methyl aspartic acid lyase shown as SEQ ID NO.1, wherein the 329 rd glutamine is mutated into arginine, the 331 th lysine is mutated into isoleucine, the 361 th cysteine is mutated into arginine, and the 365 th aspartic acid is mutated into leucine;
the mutant EcMAL-M3 is characterized in that the amino acid sequence of the mutant is that glutamine at the 329 th position of methyl aspartic acid lyase shown as SEQ ID NO.1 is mutated into histidine, lysine at the 331 st position is mutated into serine, cysteine at the 361 st position is mutated into cysteine, and aspartic acid at the 365 st position is mutated into serine;
the mutant EcMAL-M4 is characterized in that the amino acid sequence of the mutant is that glutamine at the 329 th position of methyl aspartic acid lyase shown as SEQ ID NO.1 is mutated into valine, lysine at the 331 st position is mutated into cysteine, cysteine at the 361 st position is mutated into cysteine, and aspartic acid at the 365 st position is mutated into leucine.
The amino acid sequence of the mutant EcMAL-M1 is shown as SEQ ID NO. 3;
the amino acid sequence of the mutant EcMAL-M2 is shown as SEQ ID NO. 5;
the amino acid sequence of the mutant EcMAL-M3 is shown as SEQ ID NO. 7;
the amino acid sequence of the mutant EcMAL-M4 is shown in SEQ ID NO 9.
The nucleotide sequence for coding the mutant EcMAL-M1 gene is shown as SEQ ID NO. 4;
the nucleotide sequence of the gene for coding the mutant EcMAL-M2 is shown as SEQ ID NO. 6;
the nucleotide sequence of the gene for coding the mutant EcMAL-M3 is shown as SEQ ID NO. 8;
the nucleotide sequence of the gene for coding the mutant EcMAL-M4 is shown as SEQ ID NO. 10.
The methyl aspartate lyase is derived from Escherichia coli E.coli O157: H7.
A method for preparing a mutant of the methyl aspartate lyase EcMAL comprises the following steps:
s1, connecting an EcMAL enzyme gene into a plasmid to obtain a recombinant plasmid;
s2, designing a mutation primer, carrying out PCR amplification by adopting the mutation primer and taking the recombinant plasmid as a template, carrying out full-plasmid amplification by taking a PCR amplification product as a large primer to obtain a mutation product, and transforming the mutation product into host cells to obtain a mutant library;
s3, carrying out high-throughput screening on the mutant library to obtain the methyl aspartic acid lyase EcMAL mutant.
In step S1, the nucleotide sequence of the EcMAL enzyme gene is shown as SEQ ID NO.2.
In step S1, the plasmid is preferably any one of PET-32a (+), PET-28a, PET-22b and PET-21 a.
In step S2, the mutation primers are 329-331-F and 361-365-R:
329-331-F：5’-TGTCACATGGTGNDTATCNDTACCCCGG-3’；
361-365-R：5’-GGCACTGACAHNAGTTTCATTAHNGGTGCCG-3’；
wherein NDT represents any one of twelve amino acids (R/N/D/C/G/H/I/L/F/S/Y/V).
In step S2, the large primers are:
a large primer F:
5’-TGTCACATGGTGNDTATCNDTACCCCGGATCTGGGCAGTATTCACAACATCGTCGATGCGGTTCTTTACTGCAACAGCCACAGCATGGAAGCGTACCAGGGCGGCACCNDTAATGAAACTNDTGTCAGTGCC-3’；
a large primer R:
5’-GGCACTGACAHNAGTTTCATTAHNGGTGCCGCCCTGGTACGCTTCCATGCTGTGGCTGTTGCAGTAAAGAACCGCATCGACGATGTTGTGAATACTGCCCAGATCCGGGGTAHNGATAHNCACCATGTGACA-3’。
in step S2, the host cell is preferably any one of e.coli BL21 (DE 3), e.coli BL21 (DE 3) pLysS, and Rosetta (DE 3).
The invention utilizes the high specificity of D-aspartate oxidase (D-ASPO) to oxidize D-aspartate to generate iminosuccinic acid and generate a by-product H 2 O 2 And horseradish peroxidase (HRP) in H 2 O 2 The characterisation of the colour developer 3'3 Diaminobenzidine (DAB) to a reddish brown compound in the presence of this was carried out by high throughput screening.
In step S3, the method for obtaining the mutant of the isozyme EcMAL by performing high throughput screening on the mutant library is preferably as follows: coating the mutants in the mutant library on a culture medium attached with a nylon membrane for culturing and inducing to obtain an induced nylon membrane with the mutants, performing freeze-thawing treatment on the induced nylon membrane with the mutants to obtain a freeze-thawed nylon membrane with the mutants, covering the freeze-thawed nylon membrane with the mutants on filter paper containing mixed liquor, standing, and obtaining a reddish-brown single colony which is the methyl aspartic lyase EcMAL mutant; wherein: the mixed solution is a mixed solution containing horseradish peroxidase and 3'3 Diaminobenzidine (DAB).
The mixed solution preferably consists of the following components: 500mM Tris-HCl, 20mM MgCl 2 、500mM NH 4 Cl, 10mM fumaric acid, 0.1mg/mL HRP, 0.1mM DAB and 0.5mMmg/mL of the crude enzyme solution of D-ASPO.
The D-ASPO crude enzyme solution is prepared by the following method: and (3) introducing the D-ASPO gene into escherichia coli for induced expression and separation to obtain a D-ASPO crude enzyme solution.
The gene sequence of the D-ASPO is shown as SEQ ID NO. 11.
The filter paper containing the mixed solution is obtained by immersing the filter paper in the mixed solution.
The conditions for said induction are preferably: inducing for 3-8 h at 25-35 ℃; more preferably: induction was carried out at 30 ℃ for 4h.
The conditions of the standing are preferably as follows: standing and culturing for at least 30min at the temperature of 20-40 ℃; more preferably: standing and culturing at 30 deg.C for 30min.
The mutant EcMAL of the methyl aspartate lyase is applied to the preparation of D-aspartic acid.
A method for preparing D-aspartic acid by using a methyl aspartate lyase EcMAL mutant comprises the following steps:
and (3) carrying out induced culture, separation and purification on the methyl aspartate lyase EcMAL mutant to obtain a mutant enzyme EcMAL, and adding the mutant enzyme EcMAL into the reaction solution for reaction to obtain the D-aspartic acid.
The reaction solution preferably consists of the following components: 500mM Tris-HCl, 20mM MgCl 2 、500mM NH 4 Cl, 10mM fumaric acid, pH8.5.
The reaction conditions are preferably as follows: reacting at 20-40 ℃ and 100-300 rpm for 10-30 h; more preferably: the reaction was carried out at 30 ℃ and 200rpm for 20h.
Compared with the prior art, the method has the following beneficial effects:
the invention obtains the EcMAL mutant of the methyl aspartate lyase by simultaneously carrying out mutation on the 329 th site, the 331 th site, the 361 th site and the 365 th site of the methyl aspartate lyase and combining a high-throughput screening method. The mutant changes the current situation that the prior recombinase only produces L-aspartic acid, makes the production of D-aspartic acid possible by using methyl aspartic acid lyase as a biocatalyst, and is expected to be used in the pharmaceutical industry.
Drawings
FIG. 1 is a graph showing the results of the standard mixing phase of L-aspartic acid and D-aspartic acid.
FIG. 2 is a liquid phase result chart of L-aspartic acid and D-aspartic acid produced by methyl aspartic acid lyase EcMAL produced by recombinant bacterium E.coli BL21 (DE 3)/pET-32 a-mal (EcMAL) after the end of a catalytic reaction using fumaric acid as a substrate.
FIG. 3 is a liquid phase result graph of the production of L-aspartic acid and D-aspartic acid by the mutant EcMAL produced by the mutant EcMAL-M1 after the completion of the catalytic reaction using fumaric acid as a substrate.
FIG. 4 is a liquid phase result graph of the production of L-aspartic acid and D-aspartic acid by the mutant EcMAL produced by the mutant EcMAL-M2 after the completion of the catalytic reaction using fumaric acid as a substrate.
FIG. 5 is a liquid phase result graph of the production of L-aspartic acid and D-aspartic acid by the mutant EcMAL produced by the mutant EcMAL-M3 after the completion of the catalytic reaction using fumaric acid as a substrate.
FIG. 6 is a liquid phase result graph of the production of L-aspartic acid and D-aspartic acid by the mutant EcMAL produced by the mutant EcMAL-M4 after the completion of the catalytic reaction using fumaric acid as a substrate.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Test methods without specifying specific experimental conditions in the following examples are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the invention are commercially available.
Example 1: construction of recombinant bacterium containing EcMAL
1. Coli O157H 7 genome as template, mal-F and mal-R as primers:
mal-F：5’-GCTGATATCGGATCCGAATTCATGAAAATAAAACAGGCTCTGTTCAC-3’；
mal-R：5’-GTGGTGGTGGTGGTGCTCGAGTTAATCCTTAGCCTGCAACAGCG-3’。
the mal gene in e.coli O157: H7 was amplified and EcoR I and Xho I restriction enzyme sites (underlined) were introduced at its 5 'and 3' ends, respectively. The PCR reaction system (50. Mu.L total volume) was: 2 XPrimeSTAR Max Premix 25. Mu.L, template DNA 1. Mu.L, upstream and downstream primers 1. Mu.L each, sterile water 22. Mu.L. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 5min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 80s, and 30 cycles; total extension at 72 ℃ for 10min. Obtaining PCR amplification product mal. The PCR amplification product was verified by 1% agarose gel electrophoresis, and recovered with a PCR product recovery kit (purchased from Biotechnology, shanghai, ltd.). Plasmid pET-32a (+) was subjected to a double digestion reaction with restriction enzymes EcoR I and Xho I at 37 ℃ for 4 hours, as shown in Table 1. After the enzyme digestion is finished, agarose gel electrophoresis is carried out on the enzyme digestion product, and gel recovery is carried out by using a gel recovery kit to recover the linear plasmid. The recovered linear plasmid and PCR product were mixed at a molar ratio of 1
An Ultra One Step Cloning Kit (purchased from Novowed Biotechnology Ltd.) was subjected to homologous recombination to obtain a recombinant plasmid pET-32a-mal. The recombinant plasmid is transferred into escherichia coli BL21 (DE 3) competent cells through thermal excitation to obtain a recombinant bacterium E.coli BL21 (DE 3)/pET-32 a-mal (EcMAL).
Table 1:
example 2: construction and screening of mutant libraries and validation of active mutants
(1) Construction of mutant libraries
According to the amino acid sequence of the mal gene, homologous modeling is carried out in SWISS-MODEL (https:// swissmodel. Expasy.org /), and the three-dimensional structure of EcMAL is obtained. The three-dimensional structures of fumaric acid and EcMAL are subjected to molecular docking by utilizing AutoDock Vina software, and key amino acid sites (329 th site, 331 th site and 361 th site) in the three-dimensional structures are found out. The reaction channel of the enzyme was identified and analyzed using CAVER 3.0 (http:// www. Cap. Cz /) software to determine the key amino acid positions (331 st, 365 th) near the entrance of the channel. Designing a primer (namely 329-331-F, 361-365-R) according to the key amino acid site, and carrying out PCR amplification on a gene segment obtained by amplification of the primer again to finally obtain a mutant library with four sites mutated simultaneously. The method comprises the following specific steps:
taking the recombinant plasmid pET-32a-mal as a template, and designing primers according to the key amino acid sites, wherein the primers are shown as follows:
329-331-F：5’-TGTCACATGGTGNDTATCNDTACCCCGG-3’；
361-365-R：5’-GGCACTGACAHNAGTTTCATTAHNGGTGCCG-3’；
wherein NDT represents any one of twelve amino acids (R/N/D/C/G/H/I/L/F/S/Y/V). Designing PCR amplification programs of 329-331-F and 361-365-R primers: denaturation at 95 deg.C for 3min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 10min, and 30 cycles; and carrying out total extension for 30s at 72 ℃ to obtain a gene fragment. The complementary strand of the gene fragment is used as two large primers, and the sequences are respectively as follows:
a large primer F:
5’-TGTCACATGGTGNDTATCNDTACCCCGGATCTGGGCAGTATTCACAACATCGTCGATGCGGTTCTTTACTGCAACAGCCACAGCATGGAAGCGTACCAGGGCGGCACCNDTAATGAAACTNDTGTCAGTGCC-3’；
a large primer R:
5’-GGCACTGACAHNAGTTTCATTAHNGGTGCCGCCCTGGTACGCTTCCATGCTGTGGCTGTTGCAGTAAAGAACCGCATCGACGATGTTGTGAATACTGCCCAGATCCGGGGTAHNGATAHNCACCATGTGACA-3’。
designing a large primer PCR amplification program by taking the recombinant plasmid pET-32a-mal as a template: denaturation at 95 deg.C for 3min; denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 7min for 30 cycles; total extension at 72 ℃ for 10min. Obtaining the whole plasmid PCR amplification product.
And (3) carrying out digestion treatment by using a restriction enzyme DpnI after verifying through 1% agarose gel electrophoresis, transferring the digested product into E.coli BL21 (DE 3) competent cells by using a heat shock method, and obtaining a mutant library with four sites mutated simultaneously.
(2) High throughput screening of mutant libraries
The methyl aspartic acid lyase (EcMAL) produced by the recombinant bacterium containing EcMAL catalyzes fumaric acid amination addition reaction to generate aspartic acid. The D-aspartate oxidase (D-ASPO) can be used for oxidizing D-aspartate to generate iminosuccinic acid with high specificity and generating a byproduct H 2 O 2 And horseradish peroxidase (HRP) at H 2 O 2 The characteristic that a developer 3'3 Diaminobenzidine (DAB) can be oxidized into a reddish brown compound in the presence of the reagent, a high-throughput screening method for solid-state color development was developed, comprising the following steps:
firstly, coating mutants in a mutant library on LB solid culture medium (nylon membrane is stuck on the surface of LB solid culture medium, the working concentration of ampicillin is 100 mug/mL) which is stuck with a layer of nylon membrane (purchased from general electric company in America) and has ampicillin resistance, and culturing for 16h at 37 ℃ and 180rpm overnight; transferring the nylon membrane with the mutant to an LB solid culture medium (working concentration of ampicillin is 100 mu g/mL, concentration of IPTG is 0.1 mg/mL) containing an inducer, namely isopropyl-beta-D-thiogalactoside (IPTG) and ampicillin resistance, inducing for 4h at 30 ℃ to obtain an induced nylon membrane with the mutant, transferring the nylon membrane into an empty culture dish, and repeatedly freezing and thawing for 4 times by using liquid nitrogen to obtain the frozen and thawed nylon membrane with the mutant; a piece of filter paper having the same size as the nylon membrane was dipped in the previously prepared mixture (500 mM Tris-HCl, 20mM MgCl) 2 、500mM NH 4 Cl, 10mM fumaric acid, 0.1mg/mL HRP, 0.1mM DAB, 0.5mg/mL D-ASPO crude enzyme solution); covering the freeze-thawed nylon membrane with the mutant on filter paper, and placing the filter paper in an incubator at 30 ℃ for standing for more than 30min; the single colony with reddish brown compounds is the required active mutant (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))；
Wherein: the genes for D-ASPO have been described in the articles T Shouji, T Toshiyuki, K Yoshihio et al cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humi and chromatography of the yeast enzyme DOI:10.3923/ajps.2007.399.402, and the gene sequence thereof is shown in SEQ ID NO. 11. Carrying out gene synthesis in a general biological system (Anhui) Limited company, introducing the gene into E.coli BL21 (DE 3) competent cells by taking PET-32a (+) as a carrier, culturing for 12h at 37 ℃ and 180rpm in an LB liquid culture medium (working concentration of ampicillin is 100 mug/mL) containing ampicillin resistance, transferring the inoculated amount of 1% into a fresh LB liquid culture medium containing ampicillin resistance, culturing at 37 ℃ and 180rpm, adding IPTG (isopropyl-beta-D-thiogalactoside) when the concentration of bacteria (OD 600) reaches 0.6-0.8, culturing for 8h at 25 ℃ and 180rpm, centrifuging for 5min at 4 ℃ and 8000rpm to obtain a precipitate, taking the precipitate and a Tris-HCl buffer solution (pH =8.5 and 500 mM) according to a mass ratio of 1 to 10 (g: g), carrying out ultrasonic crushing for 15min, and centrifuging for 10min at 4 ℃ and 10000rpm again to obtain a supernatant, namely a D-ASPO crude enzyme solution.
0.5mg/mL of the crude enzyme solution of D-ASPO means that 1mL of the mixed solution contains 0.5mg of the crude enzyme solution of D-ASPO.
(3) Validation of active mutants
Active mutants (i.e., mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3 and mutant EcMAL-M4) were picked and inoculated into LB liquid medium containing ampicillin resistance (working concentration of ampicillin 100. Mu.g/mL; the same applies below), culturing at 37 ℃ and 180rpm for 12h, transferring into a fresh LB liquid culture medium containing ampicillin resistance by 1% of inoculation amount, culturing at 37 ℃ and 180rpm, adding IPTG with the final concentration of 0.6-0.8 when the thallus concentration (OD 600) reaches 0.6-0.8, culturing at 16 ℃ and 180rpm for 20h, centrifuging at 4 ℃ and 8000rpm for 5min to obtain a precipitate, taking the precipitate and Tris-HCl buffer solution (pH =8.5, 100 mM) to be suspended according to the mass ratio of 1.
The resulting pure mutant enzyme EcMAL was added to 5mL of the reaction solution at a final concentration of 0.5mg/mL (the reaction solution consisted of 500mM Tris-HCl, 20mM MgCl) 2 、500mM NH 4 Cl, 10mM fumaric acid, pH 8.5), at 30 ℃ for 20 hours at 200rpm, terminating the reaction in a boiling water bath for 5min, centrifuging at 12000rpm for 10min, and measuring the concentration of D-aspartic acid by high performance liquid chromatography, and determining the optimum mutant by the amount of D-aspartic acid produced. Meanwhile, the recombinant bacterium E.coli BL21 (DE 3)/pET-32 a-mal (EcMAL) is used as a control to carry out the above test.
High performance liquid chromatography: a Chiral AAOA 5u 150 x 4.6mm chromatographic column was used, the mobile phase was: cuSO 4 The solution and isopropyl alcohol were mixed at a volume ratio of 95 (mL: mL) to obtain a mixture, wherein CuSO was added to the mixture 4 The concentration of the solution is 2mM, and the isopropanol is chromatographic grade isopropanol; the flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount is 20 mu L, and the peak appearance area of D-aspartic acid is measured under the wavelength of 254 nm.
Table 2:
a mixed standard liquid phase diagram of L-aspartic acid and D-aspartic acid is shown in FIG. 1. The cases of producing L-aspartic acid and D-aspartic acid after the catalytic reaction using fumaric acid as a substrate by using EcMAL produced by the methyl aspartate lyase EcMAL mutant (mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3 and mutant EcMAL-M4) and recombinant bacteria E.coli BL21 (DE 3)/pET-32 a-mal (EcMAL) are shown in FIGS. 2-6. As can be seen from FIG. 2, the recombinant bacterium E.coli BL21 (DE 3)/pET-32 a-mal expressed EcMAL could not detect the production of D-aspartic acid in the reaction catalyzing fumaric acid, but totally produced L-aspartic acid (the conversion rate is 85%, namely 10mM fumaric acid finally produces 8.5mM L-aspartic acid), which indicates that the methyl aspartic acid lyase (EcMAL) catalyzes the amination and addition reaction of fumaric acid to produce L-aspartic acid. As can be seen from FIGS. 3-6, the production of D-aspartic acid was detected in the reactions catalyzing fumaric acid for the mutant enzyme EcMAL expressed by the mutant EcMAL-M1, the mutant EcMAL-M2, the mutant EcMAL-M3 and the mutant EcMAL-M4 (wherein, the mutant EcMAL-M1 (FIG. 3) had a substrate of 10mM fumaric acid and a yield of converting fumaric acid into L/D-aspartic acid of 60%, the mutant EcMAL-M2 (FIG. 4) had a substrate of 10mM fumaric acid and a yield of converting fumaric acid into L/D-aspartic acid of 3.5mM, the mutant EcMAL-aspartic acid had a concentration of 2.5mM, the mutant aspartic acid had a relative yield of 58.3%, the mutant EcMA-M2 (FIG. 4) had a substrate of 10mM fumaric acid and a yield of 58%, the mutant EcMAL-aspartic acid had a substrate of 3.8mM, the mutant aspartic acid had a concentration of 2.0mM, the mutant MAL-M3, the mutant EcMAL-M4 had a substrate of 10mM, the mutant aspartic acid and a yield of 5.5.5%, the mutant aspartic acid had a substrate of 10mM, the mutant EcMAL-aspartic acid and a concentration of 3.3.3.3.3.3.3.3.3.3, the mutant aspartic acid, the concentration of D-aspartic acid was 0.5mM, so that the relative yield of L-aspartic acid was 86.8% and the relative yield of D-aspartic acid was 13.2%, see Table 2).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
<120> mutant of methyl aspartate lyase EcMAL, preparation method and application thereof
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 413
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> amino acid sequence of methyl aspartic acid 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 (Artificial Sequence)
<223> nucleotide sequence of 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 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 attaccaaag agctgacgcg cctcggttcc 900
ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960
gatgctgcca gttgtcacat ggtgcaaatc aaaaccccgg atctgggcag tattcacaac 1020
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 (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 (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 720
ggcactatcg gtctgatctt cgatatggat ccgcttcgct gtgcgcaata catcgccagc 780
ctggaaaaag aagcgcaagg cctgccgctc tacatcgaag ggccggtcga tgccggtaac 840
aagcccgatc aaattcgcct gctgaccgcg attaccaaag agctgacgcg cctcggttcc 900
ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960
gatgctgcca gttgtcacat ggtggatatc ggtaccccgg atctgggcag tattcacaac 1020
atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080
cataatgaaa ctgatgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140
atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac 1200
gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa 1242
<210> 5
<211> 413
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> amino acid sequence of mutant EcMAL-M2
<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 (Artificial Sequence)
<223> nucleotide sequence of 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 attaccaaag agctgacgcg cctcggttcc 900
ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960
gatgctgcca gttgtcacat ggtgcgtatc ataaccccgg atctgggcag tattcacaac 1020
atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080
cgtaatgaaa ctcttgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140
atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac 1200
gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa 1242
<210> 7
<211> 413
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> amino acid sequence of mutant EcMAL-M3
<400> 7
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 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 (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 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 attaccaaag agctgacgcg cctcggttcc 900
ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960
gatgctgcca gttgtcacat ggtgcatatc agtaccccgg atctgggcag tattcacaac 1020
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 (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 (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 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 attaccaaag agctgacgcg cctcggttcc 900
ggcgtgaaaa ttgtggccga tgaatggtgt aacacctacc aggatattgt tgatttcact 960
gatgctgcca gttgtcacat ggtggttatc tgtaccccgg atctgggcag tattcacaac 1020
atcgtcgatg cggttcttta ctgcaacagc cacagcatgg aagcgtacca gggcggcacc 1080
tgtaatgaaa ctcttgtcag tgcccgcacc tgtgtccacg tcgcccttgc cgctcgcccc 1140
atgcgtatgc tggtaaaacc agggatgggc tttgacgaag gcctcgatat cgtcttcaac 1200
gaaatgaatc gtactatcgc gctgttgcag gctaaggatt aa 1242
<210> 11
<211> 1113
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<223> 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 900
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 (Artificial Sequence)
<223> mal-F
<400> 12
gctgatatcg gatccgaatt catgaaaata aaacaggctc tgttcac 47
<210> 13
<211> 44
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<223> mal-R
<400> 13
gtggtggtgg tggtgctcga gttaatcctt agcctgcaac agcg 44
Claims (10)
1. The methyl aspartate lyase EcMAL mutant is characterized in that the mutant is any one of a mutant EcMAL-M1, a mutant EcMAL-M2, a mutant EcMAL-M3 and a mutant EcMAL-M4;
the mutant EcMAL-M1 is characterized in that the amino acid sequence of the mutant EcMAL-M1 is that the 329 nd glutamine of the methyl aspartic acid lyase shown as SEQ ID NO.1 is mutated into aspartic acid, the 331 st lysine is mutated into glycine, the 361 st cysteine is mutated into histidine, and the 365 th aspartic acid is mutated into aspartic acid;
the mutant EcMAL-M2 is characterized in that the amino acid sequence of the mutant is methyl aspartic acid lyase shown as SEQ ID NO.1, wherein the 329 rd glutamine is mutated into arginine, the 331 th lysine is mutated into isoleucine, the 361 th cysteine is mutated into arginine, and the 365 th aspartic acid is mutated into leucine;
the mutant EcMAL-M3 is characterized in that the amino acid sequence of the mutant is that glutamine at the 329 th position of methyl aspartic acid lyase shown as SEQ ID NO.1 is mutated into histidine, lysine at the 331 st position is mutated into serine, cysteine at the 361 st position is mutated into cysteine, and aspartic acid at the 365 st position is mutated into serine;
the mutant EcMAL-M4 is characterized in that the amino acid sequence of the mutant is that glutamine at the 329 th position of methyl aspartic acid lyase shown as SEQ ID NO.1 is mutated into valine, lysine at the 331 st position is mutated into cysteine, cysteine at the 361 st position is mutated into cysteine, and aspartic acid at the 365 st position is mutated into leucine.
2. The mutant of methyl aspartate lyase EcMAL according to claim 1,
the amino acid sequence of the mutant EcMAL-M1 is shown as SEQ ID NO. 3;
the amino acid sequence of the mutant EcMAL-M2 is shown as SEQ ID NO. 5;
the amino acid sequence of the mutant EcMAL-M3 is shown as SEQ ID NO. 7;
the amino acid sequence of the mutant EcMAL-M4 is shown in SEQ ID NO. 9.
3. The mutant of methyl aspartate lyase EcMAL according to claim 2,
the nucleotide sequence of the gene for coding the mutant EcMAL-M1 is shown as SEQ ID NO. 4;
the nucleotide sequence of the gene for coding the mutant EcMAL-M2 is shown as SEQ ID NO. 6;
the nucleotide sequence of the gene for coding the mutant EcMAL-M3 is shown as SEQ ID NO. 8;
the nucleotide sequence of the gene for coding the mutant EcMAL-M4 is shown as SEQ ID NO. 10.
4. The mutant EcMAL of methyl aspartate lyase according to claim 1, wherein said methyl aspartate lyase is derived from E.coli O157: H7.
5. The method for preparing the mutant EcMAL of methyl aspartate lyase of any one of claims 1-4, comprising the following steps:
s1, connecting an EcMAL enzyme gene into a plasmid to obtain a recombinant plasmid;
s2, designing a mutation primer, carrying out PCR amplification by adopting the mutation primer and taking the recombinant plasmid as a template, carrying out full-plasmid amplification by taking a PCR amplification product as a large primer to obtain a mutation product, and transforming the mutation product into host cells to obtain a mutant library;
s3, carrying out high-throughput screening on the mutant library to obtain the methyl aspartic acid lyase EcMAL mutant.
6. The production method according to claim 5,
in step S2, the mutation primers are 329-331-F and 361-365-R:
329-331-F：5’-TGTCACATGGTGNDTATCNDTACCCCGG-3’；
361-365-R：5’-GGCACTGACAHNAGTTTCATTAHNGGTGCCG-3’；
wherein NDT represents any one of twelve amino acids (R/N/D/C/G/H/I/L/F/S/Y/V);
in step S2, the large primers are:
and (3) a large primer F:
5’-TGTCACATGGTGNDTATCNDTACCCCGGATCTGGGCAGTATTCACAACATCGTCGATGCGGTTCTTTACTGCAACAGCCACAGCATGGAAGCGTACCAGGGCGGCACCNDTAATGAAACTNDTGTCAGTGCC-3’；
a large primer R:
5’-GGCACTGACAHNAGTTTCATTAHNGGTGCCGCCCTGGTACGCTTCCATGCTGTGGCTGTTGCAGTAAAGAACCGCATCGACGATGTTGTGAATACTGCCCAGATCCGGGGTAHNGATAHNCACCATGTGACA-3’。
7. the production method according to claim 5,
in step S3, the method for performing high throughput screening on the mutant library to obtain the isozyme EcMAL mutant comprises: coating the mutants in the mutant library on a culture medium attached with a nylon membrane for culturing and inducing to obtain an induced nylon membrane with the mutants, performing freeze-thawing treatment on the induced nylon membrane with the mutants to obtain a freeze-thawed nylon membrane with the mutants, covering the freeze-thawed nylon membrane with the mutants on filter paper containing mixed liquor, standing, and obtaining a reddish-brown single colony which is the methyl aspartic lyase EcMAL mutant; wherein: the mixed solution is a mixed solution containing horseradish peroxidase and 3'3 diaminobenzidine;
the mixed solution consists of the following components: 500mM Tris-HCl, 20mM MgCl 2 、500mM NH 4 Cl, 10mM fumaric acid, 0.1mg/mL HRP, 0.1mM DAB and 0.5mMmg/mL of the crude enzyme solution of D-ASPO;
the gene sequence of the D-ASPO is shown as SEQ ID NO. 11.
8. The method of claim 7,
the D-ASPO crude enzyme solution is prepared by the following method: introducing the D-ASPO gene into escherichia coli for induced expression and separation to obtain a D-ASPO crude enzyme solution;
the filter paper containing the mixed solution is obtained by immersing the filter paper into the mixed solution;
the induction conditions are as follows: inducing for 3-8 h at 25-35 ℃;
the standing conditions are as follows: standing and culturing for at least 30min at the temperature of 20-40 ℃.
9. Use of the methyl aspartate lyase EcMAL mutant according to any one of claims 1 to 4 for the production of D-aspartate.
10. A method for preparing D-aspartic acid by using the methyl aspartate lyase EcMAL mutant according to any one of claims 1-4, which comprises the following steps:
carrying out induction culture, separation and purification on the methyl aspartate lyase EcMAL mutant to obtain a mutant enzyme EcMAL, and adding the mutant enzyme EcMAL into a reaction solution for reaction to obtain D-aspartic acid;
the reaction solution consists of the following components: 500mM Tris-HCl, 20mM MgCl 2 、500mM NH 4 Cl, 10mM fumaric acid, pH8.5;
the reaction conditions are as follows: reacting at 20-40 deg.c and 100-300 rpm for 10-30 hr.
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