CN115851691A - Aspartic acid ammonia lyase mutant and application thereof - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to an aspartate ammonia lyase mutant and application thereof. The aspartate ammonia lyase mutant LmASpA-Mut has the ability to efficiently catalyze the hydrogenation of high-concentration acrylic acid to generate β-alanine. The wet bacterium E.coliBL21(DE3)/pET28a-LmAspA-Mut expressing the mutant LmAspA-Mut was used as a biocatalyst, and the reaction system was constructed with 315g/L acrylic acid and 125g/L ammonia as substrates, and β-alanine was obtained The yield is as high as 99%, and the product concentration is 385.5g/L.

Description

A kind of aspartic acid ammonia lyase mutant and its application

technical field

The invention relates to the field of biotechnology, in particular to an aspartate ammonia lyase mutant and application thereof.

Background technique

Beta-alanine, also known as 3-alanine, is a non-protein amino acid. β-alanine was discovered in uracil degradation products by Rose and Monroe in 1972. It is the precursor of various substances and is widely used in medicine, food, chemical industry, feed and so on. In organisms, β-alanine can regulate the metabolism in the body and act as a neurotransmitter or hormone regulator; in the field of animal breeding and production, β-alanine can improve the conversion ability of animal feed, thereby improving feed production performance; in terms of body movement, since β-alanine is the precursor required for the synthesis of carnosine in the body, supplementing an appropriate amount of β-alanine can significantly increase the content of carnosine in the muscles in the body; in the field of food, β-alanine Amino acid can be used as a flavoring agent and can effectively prevent food oxidation; in the environmental field, poly-β-alanine can be used as an efficient purification coagulant for water purification and clarification; in the medical field, the poly-β-alanine The main function is to synthesize pantothenic acid and calcium pantothenate.

There are two types of production methods for β-alanine: chemical synthesis and biosynthesis, and biosynthesis can be further divided into microbial fermentation and biocatalysis. At present, chemical synthesis is still the mainstream method in the production of β-alanine, and the more representative synthesis methods are acrylic acid method and acrylonitrile method. The chemical synthesis of β-alanine has the advantages of cheap raw materials, high yield, and mature technology. Defects such as high consumption, harsh production conditions, and more waste will have a very serious impact on the environment and do not meet the needs of green development. Therefore, there is an urgent need to establish an environmentally friendly production method for β-alanine. Biocatalysis has many advantages such as short production cycle, low production cost, less by-products, easy separation of products, and wide range of catalyst sources, and has gradually become a research hotspot in the production of β-alanine. There are two main routes for the production of β-alanine by biocatalysis, one is based on β-aminopropionitrile, and the other is based on L-aspartic acid or fumaric acid. The production route of β-alanine using β-aminopropionitrile as a substrate has a low conversion rate and is limited by the microorganism's own synthesis pathway, which will cause substrate inhibition, cannot increase the product concentration, and has many by-products. Separation and purification, not suitable for industrial production. The technological route using L-aspartic acid or fumaric acid as a substrate mainly involves L-aspartic acid-α-decarboxylase and aspartate ammonia lyase. L-aspartate-α-decarboxylase is responsible for the decarboxylation of L-aspartate to β-alanine in the biosynthetic pathway. The wild-type L-aspartate-α-decarboxylase has problems such as low activity, poor thermal stability and mechanical inactivation. Although a large number of targeted transformation results have been reported, the improvement is not large. And L-aspartic acid is used as a catalytic substrate, and the cost is relatively high.

Contents of the invention

In view of this, the present invention provides an aspartate ammonia lyase mutant and application thereof. The mutant is LmAspA-Mut, which has the ability to efficiently catalyze the hydrogenation of high-concentration acrylic acid to generate β-alanine

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

A mutant of aspartate ammonia lyase, which is obtained by mutation of at least one site among T189V, M323I, K326M and N328A of wild type aspartate ammonia lyase.

The present invention selects aspartate ammonia lyase LmAspA derived from Lysinibacillus manganicus as the research object, and successfully obtains mutants containing four site mutations through homology modeling, molecular docking and introduction of mutations. The four site mutations include T189V, At least one of M323I, K326M, N328A.

Among them, the mutant having the above four mutations is denoted as LmAspA-Mut, and its amino acid sequence is shown in SEQ ID NO.4.

The aspartate ammonia lyase LmASpA does not have the activity of catalyzing the hydrogenation of acrylic acid, while the aspartate ammonia lyase mutant LmASpA-Mut obtained by the transformation of the present invention exhibits It has a very high activity and is a biocatalyst with great application potential.

In the present invention, the amino acid sequence of the wild-type aspartate ammonia lyase is shown in SEQ ID NO.1.

The present invention also provides nucleic acid encoding the aspartic acid ammonia lyase mutant.

In some embodiments, the nucleotide sequence of the nucleic acid encoding the aspartate ammonia lyase mutant is shown in SEQ ID NO.5.

The present invention also provides a biological material comprising the nucleic acid, wherein the biological material is an expression vector or a recombinant host.

In some specific embodiments, the expression vector comprising the gene encoding the aspartate ammonia lyase mutant LmAspA-Mut provided by the present invention is obtained by the following method:

The aspartate ammonia lyase LmASpA gene shown in SEQ ID NO.2 is codon-optimized to obtain the optimized LmAspA gene. The nucleotide sequence is shown in SEQ ID NO.3, corresponding to aspartate ammonia lyase The amino acid sequence of LmAspA is shown in SEQ ID NO.1. The codon-optimized LmAspA gene (SEQ ID NO.3) was artificially synthesized and inserted between EcoRI and HindIII of pET28a to obtain the recombinant plasmid pET28a-LmAspA. Using the recombinant plasmid pET28a-LmASpA as a template, the whole plasmid was amplified by reverse PCR using primers with mutated bases, and the obtained PCR product was digested with DpnⅠ enzyme to digest the methylated template, and the digested product was transformed into Escherichia coli E.coli In BL21(DE3), the engineering bacteria E.coli BL21(DE3)/pET28a-LmAspA-Mut containing the aspartic acid ammonia lyase mutant LmASpA-Mut gene can be obtained, wherein the recombinant aspartic acid The expression vector of the gene encoding the mutant ammonia lyase was named pET28a-LmAspA-Mut.

The present invention also provides the application of the aspartate ammonia lyase mutant, the nucleic acid, or the biological material in the preparation of β-alanine.

Specifically, in the above application, the aspartate ammonia lyase mutant, the nucleic acid, or the biological material is used to catalyze the hydrogenation of acrylic acid to prepare β-alanine.

The present invention also provides a method for preparing β-alanine, comprising:

Fermenting and culturing the recombinant host expressing the aspartate ammonia lyase mutant of the present invention to obtain wet cells;

The wet bacterium or the aspartate ammonia lyase mutant of the present invention is used to catalyze the reaction of acrylic acid and ammonia water, and the reaction solution is separated and purified to obtain β-alanine.

In some embodiments, the preparation method of the wet thallus comprises: using a recombinant host expressing the aspartate ammonia lyase mutant of the present invention to prepare a seed liquid, then culturing and inducing expression to obtain an induction culture liquid, and then Centrifuge the induced culture medium, discard the supernatant, and collect the wet cells.

In some specific embodiments, the preparation method of the wet thalline comprises:

Inoculate the engineered bacteria E.coli BL21(DE3)/pET28a-LmAspA-Mut containing the gene encoding the aspartate ammonia lyase mutant into LB liquid medium containing 100 μg/mL kanamycin, and culture at 37°C for 12 hours , to obtain the seed solution, inoculate the seed solution into fresh LB liquid medium containing 100 μg/mL kanamycin at an inoculum volume concentration of 2%, cultivate at 37°C until the _OD600 is 0.5-0.7, and then add the final concentration 0.2mM IPTG, induced for 10 hours at 24°C to obtain the induction culture medium, then centrifuged the induction culture medium at 4°C and 8000rpm for 10min, discarded the supernatant, and collected the wet cells.

In the method for preparing β-alanine provided by the present invention, the reaction is: react at 25-60°C, 50-400rpm for 5h; in some specific embodiments, the reaction is: react at 40°C, 150rpm 5h. In some specific embodiments, in the reaction system, the amount of the catalyst is 40 g/L in terms of wet cells, the amount of the substrate acrylic acid is 315 g/L, and the amount of ammonia water is 125 g/L.

_In the present invention, the separation and purification of the reaction solution includes: separating and purifying the product by an organic _solvent precipitation method; Add ethanol at a ratio of 8:1, let stand at room temperature for 2 hours, discard the supernatant by suction filtration, and dry the crystallized product at 60°C to obtain β-alanine.

Compared with the prior art, the beneficial effect of the present invention is mainly reflected in that the aspartate ammonia lyase LmAspA does not have the ability to catalyze the hydrogenation of acrylic acid to generate β-alanine, and by introducing four point mutations T189V/M323I/K326M/ N328A endows the aspartate ammonia lyase mutant LmASpA-Mut with the ability to efficiently catalyze the hydrogenation of high-concentration acrylic acid to generate β-alanine. The wet bacterial cell E.coli BL21(DE3)/pET28a-LmAspA-Mut expressing the mutant LmASpA-Mut was used as a biocatalyst, and the reaction system was constructed with 315g/L acrylic acid and 125g/L ammonia water as substrates, at 40°C, 150rpm Under the conditions of reaction for 5 hours, the yield of β-alanine was as high as 99%, and the product concentration was 385.5g/L.

Description of drawings

Fig. 1 is the schematic diagram that aspartate ammonia lyase catalyzes the ammoniation of alanine to synthesize β-alanine;

Figure 2 is the agarose gel electrophoresis of the gene LmASpA encoding aspartate ammonia lyase and pET-28a; lane M is marker; lane 1 is the gene encoding aspartate ammonia lyase LmAspA; lane 2 is pET-28a ;

Fig. 3 is the agarose gel electrophoresis figure of plasmid pET28a-LmASpA after reverse PCR amplification; Swimming lane M is marker; Swimming lanes 1, 2, 3 are plasmid reverse PCR products;

Figure 4 is the SDS-PAGE image of the aspartate ammonia lyase mutant LmASpA-Mut; lane M is marker; lane 1 is produced by uninduced wet bacteria E.coli BL21(DE3)/pET28a-LmAspA-Mut The obtained crude enzyme liquid; lane 2 is the crude enzyme liquid enzyme liquid prepared by wet bacterium E.coli BL21(DE3)/pET28a-LmASpA-Mut after induction, and swimming lane 3 is the aspartate ammonia lyase mutant LmAspA -Mut separation and purification of the obtained pure enzyme liquid;

Fig. 5 is the HPLC detection spectrum of β-alanine;

Fig. 6 is the HPLC detection standard curve of β-alanine;

Fig. 7 is aspartic acid ammonia lyase mutant LmAspA-Mut catalyzed acrylic acid hydrogenation reaction process;

Fig. 8 is the HPLC detection spectrum of the beta-alanine standard substance that purity is 99%;

Fig. 9 is the HPLC detection spectrum of the obtained β-alanine separated and purified from the reaction solution;

Figure 10 is the ¹ H NMR (a) and ¹³ C NMR (b) spectra of β-alanine standard sample;

Fig. 11 is the ¹ H NMR (a) and ¹³ C NMR (b) spectra of β-alanine separated and purified from the reaction solution.

Detailed ways

The invention provides an aspartate ammonia lyase mutant and application thereof. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

Unless otherwise specified, the test materials used in the present invention are all common commercial products, which can be purchased in the market.

Below in conjunction with embodiment, further set forth the present invention:

Embodiment 1: Construction of genetically engineered bacteria E.coli BL21(DE3)/pET28a-LmASpA

The aspartate ammonia lyase LmAspA coding gene (nucleotide sequence shown in SEQ ID NO.2) derived from Lysinibacillus manganicus is codon optimized, and the nucleotide sequence of the LmASpA gene after codon optimization is shown in SEQ ID As shown in NO.3, the amino acid sequence corresponding to aspartate ammonia lyase LmASpA is shown in SEQ ID NO.1, which was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The codon-optimized LmAspA coding gene (SEQ ID NO.3) was artificially synthesized and inserted between EcoRI and HindⅢ of pET28a to obtain the recombinant plasmid pET28a-LmAspA, aspartate ammonia lyase coding gene LmASpA and pET-28a agar The sugar gel electrophoresis picture is shown in Figure 2.

Take 5 μL of the synthetic recombinant expression plasmid pET28a-LmAspA and add it to 50 μL E.coli BL21(DE3) competent medium, flick the tube wall to mix well, and place on ice for 30 minutes. Shock in a water bath at 42°C for 90 seconds, and immediately place on ice for 5 minutes. Add 900 μL LB liquid medium to the tube, and incubate at 37° C. for 1 hour on a shaker. The culture solution was centrifuged at 4000 rpm for 3 min, and 900 μL of the supernatant was taken out. Use the remaining medium to suspend the bacteria, take out 100 μL and apply it on the LB solid medium containing 100 μg/mL kanamycin. Cultivate overnight in a 37° C. incubator for 14 hours to obtain genetically engineered bacteria E.coli BL21(DE3)/pET28a-LmASpA.

LB liquid medium composition: yeast extract 5g/L, tryptone 10g/L, NaCl 10g/L, solvent is distilled water, pH 7.0-7.5.

Example 2: Construction of recombinant expression plasmid pET28a-LmASpA-Mut and genetically engineered bacteria E.coli BL21(DE3)/pET28a-LmASpA-Mut

1. Recombinant expression plasmid pET28a-LmAspA-Mut

Using the plasmid pET28a-LmAspA prepared in Example 1 as a template, primers were designed, and single-point mutations, three-point combined mutations and four-point combined mutations of key amino acids (T189/M323/K326/N328) were performed to find the best combination. The whole plasmid was cloned by inverse PCR technology and transformed into Escherichia coli E.coli BL21(DE3). Extract the plasmid for sequencing, and use software to analyze the sequencing results. The sequence contains an open reading frame with a length of 1410bp, and the 189th threonine is successfully changed to valine, and the 323rd methionine is mutated to isoleucine. acid, the 326th lysine is mutated to methionine, and the 328th asparagine is mutated to alanine. Then, the mutant plasmid was transformed into Escherichia coli E.coli BL21(DE3), the plasmid was extracted and sequenced, and the mutant plasmid pET28a-LmASpA-Mut was successfully obtained. The nucleotide sequence of the mutant LmASpA-Mut is shown in SEQ ID NO.5. The amino acid sequence is shown in SEQ ID NO.4. Each primer is as follows:

M1-F 5'-GGTCGCACTCATCTGCAGGATGCAGTTCCG-3' (SEQ ID NO. 6);

M1-R 5'-CAGATGAGTGCGACCCATTTTAATGATGCC-3' (SEQ ID NO. 7);

M2-F 5'-AGCATTATGCCGGGTATGGTTGCACCGGTT-3' (SEQ ID NO. 8);

M2-R 5'-ACCCGGCATAATGCTGCTGCCAGGCTGACG-3' (SEQ ID NO.9);

M3-F 5'-CCGGGTAAGGTTGCACCGGTTATGCCGGAA-3' (SEQ ID NO. 10);

M3-R 5'-TGCAACCTTACCCGGCATAATGCTGCTGCC-3' (SEQ ID NO. 11);

M4-F 5'-AAGGTTAATCCGGTTATGCCGGAAGTTATG-3' (SEQ ID NO. 12);

M4-R 5'-AACCGGATTAACCTTACCCGGCATAATGCT-3' (SEQ ID NO. 13);

M5-F 5'-GGACGTACACATCTTCAAGATGCTGTTCCA-3' (SEQ ID NO. 14);

M5-R 5'-AAGATGTGTACGTCCCATTTTAATAATGCC-3' (SEQ ID NO. 15);

M6-F 5'-TCAATTATGCCAGGTATGGTGGCCCCTGTT-3' (SEQ ID NO. 16);

M6-R 5'-ACCTGGCATAATTGATGAACCAGGCTGTCT-3' (SEQ ID NO. 17);

M7-F 5'-CCAGGTAAAGTGGCCCCTGTTATGCCAGAA-3' (SEQ ID NO. 18);

M7-R 5'-GGCCACTTTACCTGGCATAATTGATGAACC-3' (SEQ ID NO. 19);

M8-F 5'-AAAGTGAACCCTGTTATGCCAGAAGTTATG-3' (SEQ ID NO. 20);

M8-R 5'-AACAGGGTTCACTTTACCTGGCATAATTGA-3' (SEQ ID NO. 21);

M9-F 5'-GGACGTACACATCTTCAAGATGCTGTTCCA-3' (SEQ ID NO. 22);

M9-R 5'-AAGATGTGTACGTCCCATTTTAATAATGCC-3' (SEQ ID NO. 23).

The inverse PCR amplification system is shown in Table 1.

Table 1 PCR amplification reaction system

The PCR reaction process was as follows: pre-denaturation at 95°C for 2 min; then, complete denaturation at 95°C for 15 s, annealing at 62°C for 15 s, and extension at 72°C for 6 min. Repeat denaturation to extension steps 30 times, extend again at 72°C for 10 min, and cool down to 4°C. The PCR product was detected by 0.8% agarose gel electrophoresis. It can be seen from Figure 3 that there is a bright band at about 6500 bp, which is consistent with the theoretical value of the plasmid.

2. Genetically engineered bacteria E.coli BL21(DE3)/pET28a-LmASpA-Mut

The PCR product was digested at 37°C for 1 hour to remove the methylated template. The enzyme digestion system is shown in Table 2.

Table 2 Digestion system of methylated templates in PCR products

The PCR product after DpnI digestion (the nucleotide sequence of the mutant gene LmAspA-Mut carried by the plasmid is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.4), directly transform the expression host bacteria Escherichia coli E.coli BL21(DE3), Escherichia coli genetically engineered strain E.coli BL21(DE3)/pET28a-LmAspA-Mut was obtained. After colony PCR verification, the transformants were inserted into LB liquid medium containing 100 μg/mL kanamycin, cultured at 37°C for 12 hours, collected by centrifugation, plasmids were extracted, and sent for sequencing. The amino acid sequence of the sequencing results was analyzed by software. The 189th threonine was successfully changed to valine, the 323rd methionine was mutated to isoleucine, the 326th lysine was mutated to methionine, and the 328th lysine was mutated to methionine. Asparagine is mutated to alanine.

Example 3: Induced expression and isolation and purification of aspartic acid ammonia lyase LmASpA and its mutant LmAspA-Mut

Shake flask culture to obtain wet bacteria: Inoculate the engineered bacteria E.coliBL21(DE3)/pET28a-LmAspA-Mut containing the gene encoding the aspartate ammonia lyase mutant into LB liquid culture containing 100 μg/mL kanamycin culture medium at 37°C for 12 hours to obtain seed liquid, inoculate the seed liquid with an inoculum volume concentration of 2% into fresh LB liquid medium containing 100 μg/mL kanamycin, and cultivate at 37°C until the OD ₆₀₀ is 0.5 ~0.7, then add IPTG with a final concentration of 0.2mM, induce at 24°C for 10h, obtain the induction culture medium, then centrifuge the induction culture medium at 4°C and 8000rpm for 10min, discard the supernatant, and collect the wet cells.

Fermenter culture to obtain wet bacteria: Inoculate the genetically engineered bacteria E.coli BL21(DE3)/pET28a-LmAspA-Mut constructed in Example 2 into LB liquid medium containing 100 μg/mL kanamycin, and culture at 37°C After 12 hours, the seed solution was obtained. Operate in a clean bench, add 3mL of Kana resistance to the MgSO ₄ solution, ignite the inoculation loop, pour the MgSO ₄ /Kana solution into the medium in the fire ring, and the final concentration of kanamycin in the fermentation medium The concentration is 100 μg/mL, and the seed liquid is transferred from the fire ring to the fermenter according to the inoculum amount of 3%. Adjust the rotation speed to 600rpm, increase the ventilation rate to 6L/min, and culture at 37°C until the OD ₆₀₀ is 4-6 (about 3.5h), then add IPTG to 50mL of LB liquid medium in an ultra-clean workbench , under the protection of the fire ring, the LB medium containing IPTG was added into the fermenter through the inoculation port, and the final concentration of IPTG in the fermentation medium was 0.2mM. Reduce the temperature of the fermenter to 24°C through the circulating water system, adjust the rotation speed to 500rpm, and continue to cultivate for 10h to induce the expression of protein in the bacteria. Pour the fermented liquid into a centrifuge cup, centrifuge at 4°C at 8000 rpm for 10 min with a high-speed refrigerated centrifuge, discard the supernatant, resuspend and wash the bacterial cells with 50 mM Tris-HCl buffer (pH 8.0), and Put the bacterial suspension into a 50mL centrifuge tube, centrifuge at 12000rpm for 10min at 4°C, discard the supernatant, and collect the wet bacteria.

Add an appropriate amount of Tris-HCl (pH 8.0) buffer solution to the above-mentioned wet bacteria according to the ratio of 1g wet bacteria plus 20mL Tris-HCl buffer solution (pH 8.0), and ultrasonically break at 40% power for 15min (working 1s, intermittent 3s) , the crushed solution was centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant crude enzyme solution was obtained after repeated centrifugation three times. According to the instructions of Ni-NTA metal chelate affinity chromatography (purchased from Bio-Rad, referred to as Ni ²⁺ column, the inner diameter of the column is 1.6 cm, and the column height is 15 cm), 15 mL of the crude enzyme solution was loaded onto the pre-equilibrated Ni ²⁺ In the column, use the eluent containing 5mM imidazole, 50mM imidazole, 100mM imidazole, 200mM imidazole, 500mM imidazole successively (the eluent composition: the sodium chloride of imidazole of corresponding concentration, 300mM, the Tris-HCl of solvent is 50mM buffer, pH 8.0) to elute the impurity protein and the target protein, the elution rate is 2.5mL/min, each concentration eluent is eluted for 3 column volumes, and the effluent corresponding to the eluent containing 200mM imidazole is collected, Then use an ultrafiltration tube with a molecular weight cut-off of 10kDa to centrifuge at 4°C and 5000rpm for 30 minutes for desalination and concentration, and take the retentate, namely the LmASpA-Mut (T189V/M323I/K326M/N328A) enzyme solution, and store it at -20°C for later use.

The purity of the pure enzyme solution of the aspartate ammonia lyase mutant LmASpA-Mut was verified by SDS-PAGE gel electrophoresis, and the results of SDS-PAGE electrophoresis are shown in FIG. 4 . The aspartate ammonia lyase mutant LmAspA-Mut showed a single band after SDS-PAGE electrophoresis. The theoretical subunit size of aspartate ammonia lyase mutant LmAspA-Mut is about 52kDa, and its apparent size on SDS-PAGE electrophoresis conforms to the theoretical molecular weight.

Similarly, the wet bacterium E. coli BL21(DE3)/pET28a-LmAspA induced to express LmAspA can be obtained according to the operation procedure of Example 3, which is used as a control in the catalytic reaction of the whole cell.

Example 4: HPLC analysis of β-alanine in the process of enzymatically catalyzing the hydrogenation of acrylic acid to generate β-alanine

Mobile phase preparation: Phase A: Weigh 2.05g of anhydrous sodium acetate, pipette 1.5mL of glacial acetic acid, add 1L of ultrapure water and stir to dissolve, then filter through a 0.22μm water-based filter membrane. Phase B: Accurately measure 1L of liquid phase methanol, and filter through a 0.45μm organic filter membrane. The two phases A and B are mixed according to (v:v) 1:1, and degassed in an ultrasonic cleaner for 30 minutes, which is the mobile phase.

(250×4.6/5μm)柱；流动相流速为1mL/min；检测波长为360nm；检测温度设置为40℃。根据不同浓度的β-丙氨酸标样出峰时间，确定出β-丙氨酸的保留时间为6.194min，衍生化试剂的保留时间为4.515min(图5)。保留时间为2.713、3.148、3.293、5.822、6.589、9.668min均为衍生化反应带来的副产物峰。HPLC detection conditions: chromatographic column is Welchrom

(250×4.6/5μm) column; mobile phase flow rate is 1mL/min; detection wavelength is 360nm; detection temperature is set to 40°C. According to the elution time of the standard sample of β-alanine at different concentrations, the retention time of β-alanine was determined to be 6.194min, and the retention time of the derivatization reagent was 4.515min (Fig. 5). The retention times of 2.713, 3.148, 3.293, 5.822, 6.589, and 9.668 min are by-product peaks brought about by the derivatization reaction.

Prepare 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 M β-alanine aqueous solutions, take 100 μL respectively into 1.5mLEP tubes, add 100 μL Na ₂ CO ₃ /NaHCO ₃ buffer solution (pH 9.0), 100 μL 1% 2, 4-Dinitrofluorobenzene-acetonitrile solution was reacted in a metal bath at 60°C in the dark for 1 hour, and then 700 μL of NaH ₂ PO ₄ /Na ₂ HPO ₄ buffer solution (pH 7.0) was added. Centrifuge at 12000rpm for 5min, take the supernatant and filter it into a brown liquid phase bottle with a 0.22μm filter membrane, and detect it by HPLC to determine the peak eluting time of β-alanine and the peak area corresponding to each concentration. Plot the concentration of β-alanine and the corresponding peak area to obtain the standard curve of concentration of β-alanine and peak area, as shown in FIG. 6 . The equation of the standard curve is y=7.0407×10 ⁸ X+3.3855×10 ⁶ , y is the peak area, x is the concentration of β-alanine (in M), R ² is 0.9992, and the linear relationship is good.

The catalytic reaction solution was centrifuged at 12000rpm for 10min. Take 100 μL supernatant and add 900 μL ultrapure water, repeat once, and dilute a total of 100 times. Take 100 μL of the diluted reaction solution in a 1.5mL EP tube, add 100 μL of Na ₂ CO ₃ /NaHCO ₃ buffer solution (pH 9.0), 100 μL of 1% 2,4-dinitrofluorobenzene-acetonitrile solution, at 60°C 700 μL of NaH ₂ PO ₄ /Na ₂ HPO ₄ buffer solution (pH 7.0) was added after reacting for 1 h in a metal bath protected from light. Centrifuge at 12000rpm for 5min, take the supernatant and filter it into a brown liquid phase bottle with a 0.22μm filter membrane, and detect the peak area of β-alanine corresponding to the catalytic reaction solution of each mutant by HPLC, so as to compare the catalytic ability.

Example 5: Aspartate ammonia lyase mutant LmAspA-Mut catalyzes the hydrogenation of acrylic acid to prepare β-alanine

The wet thallus obtained by fermenting and culturing the engineering bacteria E.coli BL21(DE3)/pET28a-LmAspA-Mut containing the gene encoding the aspartate ammonia lyase mutant was used as a catalyst, and the amount of the catalyst was 40g/L, and the substrate was added 315g/L acrylic acid and 125g/L ammonia water, add water to make up the reaction system to 10mL. React the constructed 10mL catalytic reaction system at 40°C, 150rpm in a water bath shaker, take 10μL of the reaction solution every 1h and dilute it 100 times, after the DNFB derivatization reaction, detect the content of β-alanine by HPLC, according to the standard curve Calculate the concentration of β-alanine, and further calculate the product yield. The relationship between product yield and reaction time is shown in Figure 7. In a 10mL reaction system, when the reaction temperature is 40°C, the yield of β-alanine can reach 99% in 5 hours, corresponding to a product concentration of 385.5g/ L. Under the same conditions, the wet bacteria obtained from the fermentation of the engineering bacteria E.coli BL21(DE3)/pET28a-LmASpA containing the gene encoding aspartate ammonia lyase was used as a control, and no β-propane was detected in the reaction solution. Amino acid generation, indicating that it is the introduced mutation T189V/M323I/K326M/N328A that endows the aspartate ammonia lyase mutant LmASpA-Mut with the ability to efficiently catalyze the hydrogenation of high-concentration acrylic acid to generate β-alanine.

Centrifuge the reaction solution to collect the supernatant, add absolute ethanol to the supernatant according to the ratio of V _ethanol :V _{reaction supernatant} of 8:1, and let it stand at room temperature for 2 hours. The product is precipitated as prismatic crystals. After the supernatant was discarded by filtration, the crystallized product was dried at 60°C to obtain β-alanine. Dissolve 3.56 mg of β-alanine standard sample with a purity of 99% and 3 mg of purified β-alanine powder in 1 mL of ultrapure water, take 100 μL each for DNFB derivatization reaction, and perform HPLC detection, detection spectrum and results As shown in Figure 8 and Figure 9. The retention time of the β-alanine standard sample is 6.158min, and the corresponding peak area is 33744935 (Figure 8), the retention time of the purified β-alanine is 6.166min, and the corresponding peak area is 28198334 (Figure 9), The calculated purity of β-alanine is 98.17%.

Dissolve 10 mg of the β-alanine standard sample with a purity of 99% and the purified β-alanine powder in 700 μL LD ₂ O, and perform NMR detection. The detection spectra are shown in Figures 10 and 11. The ¹ H NMR spectrum of the amino acid standard sample was consistent with ^{the 13} C NMR spectrum, and the product was confirmed to be β-alanine.

The above are only preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can also be made without departing from the principle of the present invention, and these improvements and modifications should also be considered Be the protection scope of the present invention.

Claims (10)

1. An aspartate ammonia lyase mutant, characterized in that it is obtained by mutation of at least one of T189V, M323I, K326M, and N328A in wild-type aspartate ammonia lyase; The amino acid sequence of the wild-type aspartate ammonia lyase is shown in SEQ ID NO.1. 2. The aspartate ammonia lyase mutant according to claim 1, characterized in that its amino acid sequence is shown in SEQ ID NO.4. 3. A nucleic acid encoding the aspartate ammonia lyase mutant according to claim 1 or 2. 4. The nucleic acid according to claim 3, characterized in that its nucleotide sequence is as shown in SEQ ID NO.5. 5. A biological material comprising the nucleic acid according to claim 3 or 4, which is an expression vector or a recombinant host. 6. Use of the aspartate ammonia lyase mutant according to claim 1 or 2, the nucleic acid according to claim 3 or 4, or the biological material according to claim 5 in the preparation of β-alanine. 7. The application according to claim 6, characterized in that, the application is: catalytic acrylic acid hydrogenation to prepare β-alanine. 8. A method for preparing β-alanine, comprising: fermenting and culturing the recombinant host expressing the aspartate ammonia lyase mutant described in claim 1 or 2, to obtain wet bacteria; The wet bacterium or the aspartate ammonia lyase mutant described in claim 1 or 2 is used to catalyze the reaction of acrylic acid and ammonia water, and the reaction solution is separated and purified to obtain β-alanine. 9. The method according to claim 8, characterized in that, the preparation method of the wet thalline comprises: utilizing the recombinant host expressing the aspartate ammonia lyase mutant described in claim 1 or 2 to prepare the seed liquid, After culturing and inducing expression, the induction culture medium is obtained, and then the induction culture medium is centrifuged, the supernatant is discarded, and the wet bacteria are collected. 10 . The method according to claim 8 , wherein the reaction is: reacting at 40° C. and 150 rpm for 5 hours. 11 .

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