CN114107270A

CN114107270A - A L-aspartate β-decarboxylase mutant

Info

Publication number: CN114107270A
Application number: CN202111485153.6A
Authority: CN
Inventors: 周哲敏; 刘中美; 郝明珠; 朱小青; 崔睿智; 周丽; 崔文璟; 郭军玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-03-01
Anticipated expiration: 2041-12-07
Also published as: CN114107270B

Abstract

The invention discloses an L-aspartate β-decarboxylase mutant, which belongs to the technical field of enzyme engineering. The specific enzyme activity of the L-aspartate β-decarboxylase mutant E88R of the present invention is increased by 2.1 times compared with the wild type, and the specific enzyme activity reaches 481±3.7 U/mg. The mutant E88R still had 59% residual enzymatic activity at 50°C for 30 minutes, and the residual enzymatic activity was 88% and 82% at pH 7.0 and pH 8.0, respectively. Compared with the wild-type enzyme, the thermostability and pH stability of the mutant enzyme E88R are greatly improved, which is beneficial to future industrial production. At pH 7.5, the specific enzyme activity of mutant E88R reached 199±2.1 U/mg, which was 1.9 times higher than that of the wild type. Using 2.5M maleic acid as the substrate, L-alanine was catalyzed into whole cells, and the production rate reached 35.66g/(L·h), which was 1.68 times higher than that of the wild type.

Description

L-aspartic acid beta-decarboxylase mutant

Technical Field

The invention relates to an L-aspartate beta-decarboxylase mutant, belonging to the technical field of enzyme engineering.

Background

L-alanine (L-Ala) is an amino acid with important value and has wide application in daily chemical, food and medicine industries. In the food industry, L-alanine can be used as an additive to improve the utilization rate of protein in the beverage, and can also be used as a sweetener to improve the sweetness of the product; the L-alanine can also be used with sodium glutamate to prepare flavoring agent for improving food flavor. In the pharmaceutical industry, L-alanine is the synthesis of vitamin B₆And important intermediates of aminopropanol.

Currently, the industrial production of L-alanine is mainly carried out by using a biological enzyme catalysis method and a fermentation method. Although the raw materials of the fermentation method are cheap and easy to obtain, the biological enzyme catalysis method provides a good development prospect for green production due to the defects of poor optical purity of products, more byproducts, difficult extraction and the like.

At present, L-alanine is synthesized by an enzyme catalysis method, wherein aspartic acid or fumaric acid is mainly used as a catalytic substrate, and in order to further reduce the cost, a novel multi-enzyme cascade catalytic route is provided in the Chinese patent application with the application publication number of CN112941003A, and maleic acid is used as a substrate for catalysis. In the multi-enzyme cascade catalysis process, the optimum pH value of the catalysis of the maleate cis-trans isomerase MaiA is 8, the optimum pH value of the catalysis of the aspartate lyase AspA is 8.5, and the optimum pH value of the catalysis of the aspartate beta decarboxylase ASD is 5. In order to further increase the yield of L-alanine, an aspartate beta-decarboxylase which is stable and highly active in alkaline environment is needed.

Therefore, the L-aspartate beta-decarboxylase with higher enzyme activity and better enzyme stability under the alkaline condition and the method for producing L-alanine with high yield are provided, and the method has important significance for producing L-alanine by industrial application.

Disclosure of Invention

The optimum pH condition of the L-aspartate beta-decarboxylase with an amino acid sequence shown as SEQ ID NO.3 is acidic (pH 5.0), and the catalytic activity is lower under an alkaline condition (pH 7.5). The mutant E88R with improved enzyme activity and stability under alkaline condition is constructed by carrying out gene mutation on the mutant.

The first purpose of the invention is to provide an L-aspartate beta-decarboxylase mutant, and the amino acid sequence is shown as SEQ ID NO. 2.

It is a second object of the present invention to provide a gene encoding the mutant.

In one embodiment of the invention, the nucleotide sequence of the gene is as shown in SEQ ID NO. 1.

The third purpose of the invention is to provide a vector carrying the gene.

It is a fourth object of the present invention to provide a cell carrying said gene, or said vector.

The fifth purpose of the invention is to provide a genetic engineering bacterium, which takes escherichia coli as a host and expresses the L-aspartate beta-decarboxylase mutant.

In one embodiment of the invention, the genetically engineered bacterium takes escherichia coli BL21 as a host.

In one embodiment of the invention, the genetically engineered bacterium uses pET series plasmids as vectors.

In one embodiment of the invention, the vector is pET28 a.

The sixth purpose of the invention is to provide a method for improving the stability of L-aspartate beta-decarboxylase, which comprises the step of mutating the 88 th glutamic acid of the L-aspartate beta-decarboxylase with an amino acid sequence shown as SEQ ID NO.3 into arginine.

The seventh purpose of the invention is to provide a method for improving the yield of L-alanine, which takes the genetic engineering bacteria as the starting strain and maleic acid as the substrate to produce L-alanine by fermentation.

The eighth purpose of the invention is to provide a method for producing the L-aspartate beta-decarboxylase mutant, which comprises the steps of inoculating the genetic engineering bacteria into a culture medium, and culturing at 35-38 ℃ to OD₆₀₀When the temperature is 0.6-0.8 ℃, adding an inducer IPTG to induce for 16-18h at 20-22 ℃.

In one embodiment of the invention, the method is to inoculate the genetically engineered bacteria in LB culture medium containing kanamycin, and to culture the genetically engineered bacteria at 35-38 ℃ and 200r/min in a shaking way until OD is reached₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG to 0.2mM, inducing for 16-18h at 20 ℃, and expressing the L-aspartate beta-decarboxylase mutant enzyme.

In one embodiment of the present invention, the method further comprises collecting the cells of the genetically engineered bacteria, crushing the cells, collecting the supernatant, membrane-filtering the supernatant, and separating the filtrate with a His Trap HP column to obtain the L-aspartate beta-decarboxylase mutant.

The invention also provides the application of the L-aspartate beta-decarboxylase mutant, the gene, the vector, the cell or the genetic engineering bacteria in the fields of medicines, foods and chemical engineering.

The invention also provides the application of the L-aspartate beta-decarboxylase mutant, the gene, the vector, the cell or the genetic engineering bacteria in preparing products containing L-alanine.

Has the advantages that:

(1) the invention obtains the L-aspartate beta-decarboxylase mutant E88R with improved enzyme activity by constructing recombinant escherichia coli expressing the L-aspartate beta-decarboxylase mutant, and the pure enzyme activity of the mutant reaches 481 +/-3.7U/mg, which is 2.1 times higher than that of a wild type.

(2) The L-aspartate beta-decarboxylase mutant provided by the invention has high activity under the condition of pH7.5, the specific enzyme activity of the mutant is 199 +/-2.1U/mg, and the specific enzyme activity is improved by 1.9 times compared with that of wild enzyme. The residual enzyme activities of the mutant E88R at the temperature of 45 ℃ and 50 ℃ are 85% and 59%, respectively, and the residual enzyme activities are 88% and 82% under the conditions of pH 7.0 and pH 8.0, respectively. Compared with the wild enzyme, the mutant enzyme E88R has greatly improved thermal stability and pH stability.

(3) 2.5M maleic acid is used as a substrate to prepare L-alanine through whole-cell catalysis, the recombinant strain expressing the mutant enzyme E88R completes the reaction 4 hours faster than the recombinant strain expressing the wild enzyme, the production rate reaches 35.66 g/(L.h), and the production rate is improved by 1.68 times compared with the wild enzyme.

Drawings

FIG. 1: the enzyme activity histograms of the wild enzyme and the mutant enzyme E88R at pH7.5, WT is the wild enzyme.

FIG. 2: the enzyme activity curves of the wild enzyme and the mutant enzyme E88R at different pH values of 45 ℃, and WT is the wild enzyme.

FIG. 3: the enzyme activity curves of the wild enzyme and the mutant enzyme E88R at different temperatures of pH 5.0, and WT is the wild enzyme.

FIG. 4: thermostability curves for the wild enzyme and the mutant enzyme E88R, WT being the wild enzyme.

FIG. 5: pH stability curves for the wild enzyme and the mutant enzyme E88R, WT being the wild enzyme.

FIG. 6: whole cell catalysis 2.5M maleic acid results; wild type E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lMutant E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-l-ASD/E88R-MaiA。

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, the reagents and materials used in the following examples are all commercially available or may be prepared by known methods.

Method for determining enzyme activity and enzymological property of L-aspartate beta-decarboxylase

(1) The method for measuring the enzyme activity of the L-aspartic acid beta-decarboxylase comprises the following steps:

at 45 ℃ and pH 5.0, the enzyme amount required for converting a substrate to generate 1 mu mol of L-Ala per minute is defined as an enzyme activity unit of 1U, and the enzyme activity unit contained in each mg of protein is defined as specific enzyme activity.

Adding a proper amount of enzyme solution 3-5 mu g, 10mM phosphate-citric acid buffer solution pH5, 0.5mM PLP and 1mM alpha-keto into a 1mL system, uniformly mixing, preheating at 45 ℃ for 5min, adding 0.2M substrate sodium aspartate, carrying out water bath reaction at 45 ℃ for 10min, and inactivating at 100 ℃ for 10min to terminate the reaction. Centrifuging at 12000rpm for 3min, and collecting supernatant to derive and detect the yield of L-alanine. The reaction mixture was filtered through a 0.22 μm microporous membrane and loaded onto a C18 column for HPLC analysis.

(2) Determination of optimum reaction pH: wild-type enzyme was used as a control. Keeping other reaction conditions unchanged, adding phosphoric acid-citric acid buffer solutions with different pH values into a reaction system, reacting at 45 ℃ for 10min, inactivating to terminate the reaction, and detecting the optimal reaction pH.

(3) Determination of optimum reaction temperature: wild-type enzyme was used as a control. Keeping other reaction conditions unchanged, carrying out reaction for 10min at different temperatures, then inactivating and terminating the reaction, and detecting the optimal reaction temperature.

(4) Determination of temperature stability: wild-type enzyme was used as a control. Placing equal amount of enzyme solution in water bath kettle at 0, 25, 45, and 55 deg.C, incubating for 30min, and detecting residual activity of enzyme for thermal stability determination.

(5) Determination of pH stability: wild-type enzyme was used as a control. Diluting the same amount of enzyme solution with buffer solution with the same volume and different pH, keeping the temperature on ice for more than 12h, keeping other reaction conditions unchanged, detecting the residual activity of the enzyme and carrying out pH stability determination.

(II) culture Medium

LB medium (g/L): 10.0 parts of peptone, 5.0 parts of yeast powder and 10.0 parts of NaCl.

2YT medium (g/L): peptone 16.0, yeast powder 10.0 and NaCl 5.0.

(III) HPLC (high Performance liquid chromatography) method for detecting contents of sodium L-aspartate and L-alanine

The reaction solution is derived by using benzene isothiocyanate (PITC), and the specific steps are as follows: adding 500 mu L of reaction solution into a 2.0mL centrifuge tube, adding 250 mu L of 0.1 mol/LPITC-acetonitrile solution and 250 mu L of 1mol/L triethylamine-acetonitrile solution, fully and uniformly mixing, standing at room temperature in a dark place for 45min, adding 750 mu L of n-hexane solution to terminate derivatization, oscillating for 1min by a vortex oscillator, standing for 30-60min, absorbing the lower layer solution, filtering by a 0.22 mu m organic filter membrane, and then injecting a sample, wherein the sample injection amount is 10 mu L.

The derivatized product was determined by HPLC: the column was La Chrom C18(5 μm, 4.6X 250 mm); the mobile phase A solution is 80 percent (V/V) acetonitrile water solution, and the B solution is 97:3(V/V, pH 6.5) 0.1mol/L sodium acetate-acetonitrile solution; gradient elution was used: the solution B is reduced from 95% to 65% in 0-20 min; after 20-30min, the liquid B is increased from 65% to 95%; 30-35min, and the gradient of the solution B is unchanged. The detection wavelength was 254nm and the column temperature was 40 ℃.

(IV) plasmids and strains

pET28a-Pd21192 ASD: has been disclosed in the patent publication No. CN112941003A and is named pET28a-ASD-2, which is self-named pET28a-Pd21192ASD in the present invention.

E.coli BL21(DE3)ΔfumAC-T7-RBS/AspA/pRSF_Duet-l-ASD-MaiA: is disclosed in publication No. CN 112941003A.

Coli BL21(DE3) Δ fumAC-T7-RBS/AspA: is disclosed in publication No. CN 112941003A.

pRSF_Duet-l-ASD-MaiA: is disclosed in publication No. CN 112941003A.

Example 1: construction and purification of recombinant Escherichia coli BL21/pET28a-E88R

(1) Construction of mutant BL21/pET28 a-E88R:

using pET28a-Pd21192ASD plasmid as a template, whole plasmid PCR was performed using E88R-UP/DOWN primers, the sequence information of which is shown in Table 1, and PCR was performed under the conditions shown in Table 2. The obtained PCR product is identified by an agarose gel electrophoresis method, and then after the PCR product is purified and digested, Escherichia coli E.coli JM109 is transformed, and plasmids are extracted to obtain a recombinant plasmid pET28a-E88R carrying the gene encoding the mutant. The recombinant plasmid pET28a-E88R is transformed into an Escherichia coli E.coli BL21 strain to obtain a recombinant strain BL21/pET28 a-E88R.

TABLE 1 primers

Name (R)	Sequence (5 '-3')
		E88R-UP	CATAgaagggcgcttcAGgcgctatattgccg
E88R-DOWN	cccttctatgccctcgatctttg

TABLE 2 Whole plasmid PCR amplification reaction System

Reagent	Dosage (mu L)
		Primerstar polymerase	25
Form panel	1.0
		Upstream primer	1.0
DownstreamPrimer and method for producing the same	1.0
		ddH₂O	22
General assembly	50

The PCR amplification reaction conditions are as follows:

(2) the recombinant strain BL21/pET28a-E88R in step (1) was inoculated in 5mL of LB medium (kanamycin concentration 50. mu.g/mL), and cultured overnight at 37 ℃ with shaking at 200r/min to obtain a seed solution. The seed solution was inoculated into 100mL of LB medium (kanamycin concentration 50. mu.g/mL) at an inoculum size of 1% (v/v), and cultured at 37 ℃ with shaking at 200r/min to OD₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG to 0.2mM, inducing at 30 ℃ for 16-18h to obtain bacterial liquid, and centrifuging at 6000rpm for 10min to collect thalli cells.

(3) Resuspending the bacterial cells in step (2) with a Binding buffer, ultrasonically disrupting the cells on ice for 30min, centrifuging the disrupted cells, collecting the supernatant, filtering the supernatant with a 0.22 μm filter membrane to obtain a crude enzyme solution, and purifying the crude enzyme solution with a His Trap FF purification column. The collected purified protein is put into 50mM phosphate buffer solution with pH 6.5 for dialysis for 8-12h at 4 ℃, and the dialysate can be changed for a plurality of times during the dialysis, so that imidazole can be removed more thoroughly to obtain pure enzyme solution. SDS-PAGE gel electrophoresis was performed to check the purity of the purified protein. The measured specific enzyme activity of the pure enzyme of the mutant E88R reaches 481 +/-3.7U/mg, which is 2.1 times higher than that of the wild type.

Example 2: determination of optimum pH

Pure enzyme solutions of the mutant and wild-type enzymes in example 1 were diluted to a final concentration of 5ug/mL, with the wild-type enzyme as a control; keeping other reaction conditions unchanged, adding phosphoric acid-citric acid buffer solutions (pH 3-8) with different pH values into a reaction system, reacting at 45 ℃ for 10min, inactivating to terminate the reaction, and detecting the optimal reaction pH. As shown in FIG. 1, under the condition of pH7.5, the specific enzyme activity of the wild type is 106 +/-2.7U/mg, and the specific enzyme activity of the mutant is 199 +/-2.1U/mg, which is improved by 1.9 times compared with the wild type.

As shown in FIG. 2, the optimum reaction pH of the mutant E88R was 4.5, and the relative enzyme activity was maintained at about 60% at pH7.5, with the highest enzyme activity of each of the mutant and wild enzymes being 100%. Compared with wild type, the mutant has certain improvement on enzyme activity under acidic condition and alkaline condition.

Example 3: determination of optimum temperature

Pure enzyme solutions of the mutant and wild-type enzymes in example 1 were diluted to a final concentration of 5ug/mL, with the wild-type enzyme as a control; keeping other reaction conditions unchanged, carrying out reaction at different temperatures for 10min, then inactivating to terminate the reaction (the temperature is set to be 35-60 ℃), and detecting the optimal reaction temperature.

The results are shown in fig. 3, and the maximum enzyme activity of each of the mutant and the wild enzyme is 100%, the relative enzyme activity of the mutant is the highest at 55 ℃, 96% of the relative enzyme activity can be maintained at 60 ℃, and the relative enzyme activity of the wild enzyme is only 12%.

Example 4: determination of thermal stability

The pure enzyme solutions of the mutant and the wild-type enzyme in example 1 were diluted to a final concentration of 5ug/mL, and the wild-type enzyme was used as a control, 50 μ g of the pure enzyme solution of the mutant in example 1 was added to 500 μ L of the buffer reaction system, and incubated in a 0, 25, 45, and 55 ℃ water bath for 30min, followed by ice bath for 2min, substrate 1M L-aspartic acid 200 μ L was added, and the reaction was performed at 25, 45, and 50 ℃ for 10min to determine the residual activity of the enzyme.

The results are shown in FIG. 4, the residual enzyme activities of the mutant enzyme E88R at 45 ℃ and 50 ℃ are 85% and 59%, respectively; the residual enzyme activities of the wild enzyme at 45 ℃ and 50 ℃ are 74% and 34%, respectively.

Example 5: determination of pH stability

Wild-type enzyme was used as a control. Diluting the pure enzyme solution of the same amount of the mutant and the wild enzyme to the same concentration by using buffer solution with the same volume and different pH values, preserving the temperature for more than 12h on ice, keeping other reaction conditions unchanged, and detecting the residual activity of the enzyme to carry out pH stability determination.

The results are shown in FIG. 5, the residual enzyme activity of the mutant enzyme is 88% and 82% under the conditions of pH 7.0 and pH 8.0, respectively; the residual enzyme activity of the wild enzyme under the conditions of pH 7.0 and pH 8.0 is 78% and 36%, respectively. Under the condition of pH7.5, the specific enzyme activity of the mutant is 199 +/-2.1U/mg, the specific enzyme activity is improved by 1.9 times compared with that of the wild enzyme, and the stability of the mutant enzyme is greatly improved.

Example 6: recombinant wild strain whole cell converting maleic acid to produce L-alanine

(1) Coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lthe-ASD-MaiA was inoculated into 5mL of LB medium (kanamycin concentration 50. mu.g/mL), and cultured overnight at 37 ℃ with shaking at 200r/min to obtain a seed solution.

(2) Inoculating the seed solution of step (1) in 100mL LB medium (kanamycin concentration 50. mu.g/mL) at an inoculum size of 1% (v/v), culturing at 37 ℃ with shaking at 200r/min to OD₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG to 0.2mM, inducing at 30 ℃ for 16-18h to obtain bacterial liquid, and centrifuging at 6000rpm for 10min to collect thalli cells.

(3) Firstly, preparing a maleic acid solution with the pH of 7.5, and adjusting the pH of the maleic acid to 7.5 by using ammonia water in the process. Resuspending the bacterial cells in step (2) with 50mM phosphate buffer solution with pH7.5, diluting to obtain bacterial solution with OD600 of 20, mixing the bacterial solution with 20% (v/v) and 80% (v/v) substrate maleic acid, wherein the final concentration of maleic acid is 2.5M, the reaction system is 50mL, catalyzing the reaction in a shaker at 37 ℃ and 200r/min, and sampling every 2h to detect the contents of maleic acid, fumaric acid, L-aspartic acid and L-alanine in the reaction solution.

The results are shown in FIG. 6(A), E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lThe ASD-MaiA strain can be completely transformed in 10 hours under the catalysis of 2.5M substrate maleic acid, the transformation rate reaches over 98 percent, the yield reaches 212.7g/L, and the production rate reaches 21.27 g/(L.h).

Example 7: recombinant mutant strain whole cell converting maleic acid to produce L-alanine

(1) Mutant pRSF_Duet-lConstruction of ASD/E88R-MaiA:

with pRSF_Duet-lThe ASD-MaiA plasmid was used as a template, and whole plasmid PCR was carried out using E88R-UP/DOWN primers whose sequence information is shown in Table 1 and PCR was carried out under the conditions shown in Table 2, respectively. Identifying the obtained PCR product by agarose gel electrophoresis, purifying and digesting the PCR product, transforming E.coli JM109, extracting plasmid to obtain recombinant plasmid pRSF carrying coding mutant gene_Duet-lASD/E88R-MaiA. Recombinant plasmid pRSF_Duet-lASD/E88R-MaiA transformation E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA strain to obtain recombinant strain E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-l-ASD/E88R-MaiA。

(2) Coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lASD/E88R-MaiA was inoculated in 5mL of LB medium (kanamycin concentration 50. mu.g/mL), and cultured overnight at 37 ℃ with shaking at 200r/min to obtain a seed liquid.

(3) Inoculating the seed solution of step (2) in 100mL LB medium (kanamycin concentration 50. mu.g/mL) at an inoculum size of 1% (v/v), culturing at 37 ℃ with shaking at 200r/min to OD₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG to 0.2mM, inducing at 30 ℃ for 16-18h to obtain bacterial liquid, and centrifuging at 6000rpm for 10min to collect thalli cells.

(4) A solution of maleic acid at pH7.5 was prepared, and the pH of the maleic acid was adjusted to 7.5 with aqueous ammonia. Collecting the thallus cells induced and expressed in the step (3), re-suspending the thallus cells in the step (3) by using 50mM phosphate buffer solution with the pH of 7.5, diluting the thallus cells into a bacteria solution with the OD600 of 20, then mixing the bacteria solution with 20% (v/v) and a substrate maleic acid of 80% (v/v), wherein the final concentration of the maleic acid is 2.5M, the reaction system is 50mL, catalyzing the reaction in a shaking table at the temperature of 37 ℃ at 200r/min, and sampling every 2h to detect the contents of the maleic acid, the fumaric acid, the L-aspartic acid and the L-alanine in the reaction solution.

The results are shown in FIG. 6(B), E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lThe ASD/E88R-MaiA strain can be completely transformed in 6h under the catalysis of 2.5M substrate maleic acid, and is better than wildThe reaction is completed 4h faster than the wild type, the conversion rate reaches more than 98%, the yield reaches 213.9g/L, the production rate reaches 35.66 g/(L.h), and the yield is improved by 1.68 times compared with the wild type.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> an L-aspartate beta-decarboxylase mutant

<130> BAA211533A

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1602

<212> DNA

<213> Artificial sequence

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atgagcaagg attatcagag tctggcgaac ttgagcccgt ttgagctcaa ggatgagttg 60

atcaagatcg cctcgggcga cggaaaccgc ctcatgctca atgcggggcg gggcaatccc 120

aattttctgg caaccacccc gagaagagca tttttccgtc tgggcttgtt cgcggctgcc 180

gagtcggaac tttcgtattc atatatgaac acggtgggcg tgggaggcct ggcaaagatc 240

gagggcatag aagggcgctt caggcgctat attgccgaga accgcgatca ggaaggcgtg 300

cgctttctcg gtaaatccct gagttatgta cgcgatcagc tgggcttgga tccggccgcc 360

ttcctgcacg agatggtcga cggtattctg ggctgcaatt accccgttcc ccctcggatg 420

ctgaacatca gcgaaaaaat cgtgcgccag tacatcatcc gtgaaatggg ggccgatgca 480

attcccagcg agtccgtgaa cctgtttgcg gtcgaggggg gaacggccgc catggcatac 540

atcttcgaga gcatgaaggt caacggcctc ctcaaggctg gtgacaaggt agccatcggc 600

atgccggttt tcactccgta catagaaatt ccggaactgg cccagtatgc gttggaggag 660

gtggcaatca atgccgaccc ggccctcaac tggcaatatc ctgattccga actagacaag 720

ctcaaggatc cggccatcaa gatcttcttc tgcgtgaacc ccagcaatcc gccatcggta 780

aagatggacg agcgcagcct ggagcgtgtg cgcaagattg tggcagagca tcgaccggat 840

ctgatgatcc tgaccgatga cgtctatggc acgtttgccg atggctttca gtcgctcttt 900

gcgatttgcc cggccaacac tttgttggtc tattcattct ccaaatactt tggtgccact 960

ggctggcgtc tgggtgtcgt ggccgcccat aaggaaaata tcttcgactt ggcattgggc 1020

aggctgcctg agtccgagaa aacagcgctc gatgatcgct atcgttcact gctacccgat 1080

gtgcgttcat tgaaattcct agatcgtctg gttgccgaca gccgcgctgt tgccttgaac 1140

cacacggccg gtctgtccac gccgcagcag gtccagatga ccttgttctc gttgtttgcg 1200

ctcatggacg agagcgacca gtacaagcac acgctcaagc aactgatacg acgtcgtgaa 1260

gcaacgctct atcgcgagtt gggaacgcct ccgcaaagag atgaaaatgc ggtcgattac 1320

tacaccttga ttgacctgca ggacgtgacg tcgaagcttt atggcgaagc gttctcgaaa 1380

tgggcagtca agcagtcctc gaccggcgac atgctgttcc ggattgccga cgaaacaggg 1440

atcgtgctcc tgccgggacg tggctttgga tcggaccgtc catcgggacg cgcctccttg 1500

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<210> 2

<211> 533

<212> PRT

<213> Artificial sequence

<400> 2

Met Ser Lys Asp Tyr Gln Ser Leu Ala Asn Leu Ser Pro Phe Glu Leu

1 5 10 15

Lys Asp Glu Leu Ile Lys Ile Ala Ser Gly Asp Gly Asn Arg Leu Met

20 25 30

Leu Asn Ala Gly Arg Gly Asn Pro Asn Phe Leu Ala Thr Thr Pro Arg

35 40 45

Arg Ala Phe Phe Arg Leu Gly Leu Phe Ala Ala Ala Glu Ser Glu Leu

50 55 60

Ser Tyr Ser Tyr Met Asn Thr Val Gly Val Gly Gly Leu Ala Lys Ile

65 70 75 80

Glu Gly Ile Glu Gly Arg Phe Arg Arg Tyr Ile Ala Glu Asn Arg Asp

85 90 95

Gln Glu Gly Val Arg Phe Leu Gly Lys Ser Leu Ser Tyr Val Arg Asp

100 105 110

Gln Leu Gly Leu Asp Pro Ala Ala Phe Leu His Glu Met Val Asp Gly

115 120 125

Ile Leu Gly Cys Asn Tyr Pro Val Pro Pro Arg Met Leu Asn Ile Ser

130 135 140

Glu Lys Ile Val Arg Gln Tyr Ile Ile Arg Glu Met Gly Ala Asp Ala

145 150 155 160

Ile Pro Ser Glu Ser Val Asn Leu Phe Ala Val Glu Gly Gly Thr Ala

165 170 175

Ala Met Ala Tyr Ile Phe Glu Ser Met Lys Val Asn Gly Leu Leu Lys

180 185 190

Ala Gly Asp Lys Val Ala Ile Gly Met Pro Val Phe Thr Pro Tyr Ile

195 200 205

Glu Ile Pro Glu Leu Ala Gln Tyr Ala Leu Glu Glu Val Ala Ile Asn

210 215 220

Ala Asp Pro Ala Leu Asn Trp Gln Tyr Pro Asp Ser Glu Leu Asp Lys

225 230 235 240

Leu Lys Asp Pro Ala Ile Lys Ile Phe Phe Cys Val Asn Pro Ser Asn

245 250 255

Pro Pro Ser Val Lys Met Asp Glu Arg Ser Leu Glu Arg Val Arg Lys

260 265 270

Ile Val Ala Glu His Arg Pro Asp Leu Met Ile Leu Thr Asp Asp Val

275 280 285

Tyr Gly Thr Phe Ala Asp Gly Phe Gln Ser Leu Phe Ala Ile Cys Pro

290 295 300

Ala Asn Thr Leu Leu Val Tyr Ser Phe Ser Lys Tyr Phe Gly Ala Thr

305 310 315 320

Gly Trp Arg Leu Gly Val Val Ala Ala His Lys Glu Asn Ile Phe Asp

325 330 335

Leu Ala Leu Gly Arg Leu Pro Glu Ser Glu Lys Thr Ala Leu Asp Asp

340 345 350

Arg Tyr Arg Ser Leu Leu Pro Asp Val Arg Ser Leu Lys Phe Leu Asp

355 360 365

Arg Leu Val Ala Asp Ser Arg Ala Val Ala Leu Asn His Thr Ala Gly

370 375 380

Leu Ser Thr Pro Gln Gln Val Gln Met Thr Leu Phe Ser Leu Phe Ala

385 390 395 400

Leu Met Asp Glu Ser Asp Gln Tyr Lys His Thr Leu Lys Gln Leu Ile

405 410 415

Arg Arg Arg Glu Ala Thr Leu Tyr Arg Glu Leu Gly Thr Pro Pro Gln

420 425 430

Arg Asp Glu Asn Ala Val Asp Tyr Tyr Thr Leu Ile Asp Leu Gln Asp

435 440 445

Val Thr Ser Lys Leu Tyr Gly Glu Ala Phe Ser Lys Trp Ala Val Lys

450 455 460

Gln Ser Ser Thr Gly Asp Met Leu Phe Arg Ile Ala Asp Glu Thr Gly

465 470 475 480

Ile Val Leu Leu Pro Gly Arg Gly Phe Gly Ser Asp Arg Pro Ser Gly

485 490 495

Arg Ala Ser Leu Ala Asn Leu Asn Glu Tyr Glu Tyr Ala Ala Ile Gly

500 505 510

Arg Ala Leu Arg Gln Met Ala Asp Glu Leu Tyr Ala Gln Tyr Thr Gln

515 520 525

Gln Gly Asn Lys Arg

530

<210> 3

<211> 533

<212> PRT

<213> Artificial sequence

<400> 3

Met Ser Lys Asp Tyr Gln Ser Leu Ala Asn Leu Ser Pro Phe Glu Leu

1 5 10 15

Lys Asp Glu Leu Ile Lys Ile Ala Ser Gly Asp Gly Asn Arg Leu Met

20 25 30

Leu Asn Ala Gly Arg Gly Asn Pro Asn Phe Leu Ala Thr Thr Pro Arg

35 40 45

Arg Ala Phe Phe Arg Leu Gly Leu Phe Ala Ala Ala Glu Ser Glu Leu

50 55 60

Ser Tyr Ser Tyr Met Asn Thr Val Gly Val Gly Gly Leu Ala Lys Ile

65 70 75 80

Glu Gly Ile Glu Gly Arg Phe Glu Arg Tyr Ile Ala Glu Asn Arg Asp

85 90 95

Gln Glu Gly Val Arg Phe Leu Gly Lys Ser Leu Ser Tyr Val Arg Asp

100 105 110

Gln Leu Gly Leu Asp Pro Ala Ala Phe Leu His Glu Met Val Asp Gly

115 120 125

Ile Leu Gly Cys Asn Tyr Pro Val Pro Pro Arg Met Leu Asn Ile Ser

130 135 140

Glu Lys Ile Val Arg Gln Tyr Ile Ile Arg Glu Met Gly Ala Asp Ala

145 150 155 160

Ile Pro Ser Glu Ser Val Asn Leu Phe Ala Val Glu Gly Gly Thr Ala

165 170 175

Ala Met Ala Tyr Ile Phe Glu Ser Met Lys Val Asn Gly Leu Leu Lys

180 185 190

Ala Gly Asp Lys Val Ala Ile Gly Met Pro Val Phe Thr Pro Tyr Ile

195 200 205

Glu Ile Pro Glu Leu Ala Gln Tyr Ala Leu Glu Glu Val Ala Ile Asn

210 215 220

Ala Asp Pro Ala Leu Asn Trp Gln Tyr Pro Asp Ser Glu Leu Asp Lys

225 230 235 240

Leu Lys Asp Pro Ala Ile Lys Ile Phe Phe Cys Val Asn Pro Ser Asn

245 250 255

Pro Pro Ser Val Lys Met Asp Glu Arg Ser Leu Glu Arg Val Arg Lys

260 265 270

Ile Val Ala Glu His Arg Pro Asp Leu Met Ile Leu Thr Asp Asp Val

275 280 285

Tyr Gly Thr Phe Ala Asp Gly Phe Gln Ser Leu Phe Ala Ile Cys Pro

290 295 300

Ala Asn Thr Leu Leu Val Tyr Ser Phe Ser Lys Tyr Phe Gly Ala Thr

305 310 315 320

Gly Trp Arg Leu Gly Val Val Ala Ala His Lys Glu Asn Ile Phe Asp

325 330 335

Leu Ala Leu Gly Arg Leu Pro Glu Ser Glu Lys Thr Ala Leu Asp Asp

340 345 350

Arg Tyr Arg Ser Leu Leu Pro Asp Val Arg Ser Leu Lys Phe Leu Asp

355 360 365

Arg Leu Val Ala Asp Ser Arg Ala Val Ala Leu Asn His Thr Ala Gly

370 375 380

Leu Ser Thr Pro Gln Gln Val Gln Met Thr Leu Phe Ser Leu Phe Ala

385 390 395 400

Leu Met Asp Glu Ser Asp Gln Tyr Lys His Thr Leu Lys Gln Leu Ile

405 410 415

Arg Arg Arg Glu Ala Thr Leu Tyr Arg Glu Leu Gly Thr Pro Pro Gln

420 425 430

Arg Asp Glu Asn Ala Val Asp Tyr Tyr Thr Leu Ile Asp Leu Gln Asp

435 440 445

Val Thr Ser Lys Leu Tyr Gly Glu Ala Phe Ser Lys Trp Ala Val Lys

450 455 460

Gln Ser Ser Thr Gly Asp Met Leu Phe Arg Ile Ala Asp Glu Thr Gly

465 470 475 480

Ile Val Leu Leu Pro Gly Arg Gly Phe Gly Ser Asp Arg Pro Ser Gly

485 490 495

Arg Ala Ser Leu Ala Asn Leu Asn Glu Tyr Glu Tyr Ala Ala Ile Gly

500 505 510

Arg Ala Leu Arg Gln Met Ala Asp Glu Leu Tyr Ala Gln Tyr Thr Gln

515 520 525

Gln Gly Asn Lys Arg

530

Claims

1. A L-aspartate beta-decarboxylase mutant, characterized in that the amino acid sequence is as shown in SEQ ID NO.2.

2. A gene encoding the L-aspartate β-decarboxylase mutant of claim 1.

3. A vector carrying the gene of claim 2.

4. A cell carrying the gene of claim 2 or the vector of claim 3.

5. A genetically engineered bacterium, characterized in that, using Escherichia coli as a host, the L-aspartate β-decarboxylase mutant of claim 1 is expressed.

6 . The genetically engineered bacteria according to claim 5 , wherein the Escherichia coli BL21 is used as the host, and the pET series plasmid is used as the carrier. 7 .

7. a method for improving the stability of L-aspartic acid β-decarboxylase, it is characterized in that, by amino acid sequence such as the 88th position valley of L-aspartic acid β-decarboxylase shown in SEQ ID NO.3 amino acid to arginine.

8. a method for improving L-alanine output, is characterized in that, with the described genetically engineered bacteria of claim 5 or 6 as fermentation strain, with maleic acid as substrate, fermentation produces L-alanine.

9. A method for producing the L-aspartate β-decarboxylase mutant according to claim 1, wherein the genetically engineered bacteria according to claim 5 or 6 are inoculated into a culture medium, and the bacterium is inoculated at 35- Incubate at 38°C until OD ₆₀₀ is 0.6-0.8, add inducer IPTG to induce 16-18h at 30°C.

10. The L-aspartate β-decarboxylase mutant of claim 1, or the gene of claim 2, or the vector of claim 3, or the cell of claim 4, or claim 5 or 6 The application of the genetically engineered bacteria in the preparation of L-alanine-containing products.