Aspartate ammonia lyase mutant and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to an aspartate ammonia lyase mutant and its application. The aspartate ammonia lyase mutant LmAspA-Mut has the ability to efficiently catalyze the addition of ammonia to high-concentration acrylic acid to generate β-alanine. Using wet bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut expressing the mutant LmAspA-Mut as a biocatalyst, 315 g / L acrylic acid and 125 g / L ammonia water as substrates to construct a reaction system, the β-alanine yield is as high as 99%, and the product concentration is 385.5 g / L.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an aspartate ammonia lyase mutant and an application thereof. Background Art

[0002] β-alanine, also known as 3-aminopropionic acid, is a non-protein amino acid. β-alanine was discovered by Rose and Monroe in 1972 in the degradation products of uracil. It is a precursor of many substances and is widely used in medicine, food, chemical industry, feed, etc. In the body, β-alanine can regulate metabolism in the body and play the role of neurotransmitter or hormone regulator; in the field of animal breeding and production, β-alanine can improve the conversion capacity of animal feed, thereby improving feed production performance; in terms of body movement, since β-alanine is a 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 body's muscles; in the field of food, β-alanine can be used as a seasoning and can effectively prevent food oxidation; in the field of environment, poly-β-alanine can be used as a high-efficiency purification coagulant for water purification and clarification; in the field of medicine, the main function of β-alanine is to synthesize pantothenic acid and calcium pantothenate.

[0003] There are two types of β-alanine production methods: chemical synthesis and biosynthesis. Biosynthesis can be divided into microbial fermentation and biocatalysis. At present, chemical synthesis is still the mainstream method for β-alanine production, among which the more representative synthesis methods are acrylic acid method and acrylonitrile method. Chemical synthesis of β-alanine has the advantages of cheap raw materials, high yield, mature technology and process, but chemical synthesis of β-alanine often requires a high temperature and high pressure environment, or requires the participation of strong acid and strong base, and has defects such as high energy consumption, harsh production conditions, and more waste, which will have a very serious impact on the environment and does not meet the needs of green development. Therefore, it is urgent to establish an environmentally friendly β-alanine production method. Biocatalysis has many advantages such as short production cycle, low production cost, fewer by-products, easy product separation, and a wide range of catalyst sources. It has gradually become a hot research topic in the production of β-alanine. There are two main process routes for the production of β-alanine by biocatalysis, one is to use β-aminopropionitrile as a substrate, and the other is to use L-aspartic acid or fumaric acid as a substrate. The process route for producing 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 produce substrate inhibition and cannot increase the product concentration. In addition, there are many by-products that are difficult to separate and purify, making it unsuitable for industrial production. The process route using L-aspartate or fumarate as a substrate mainly involves L-aspartate-α-decarboxylase and aspartate ammonia lyase. L-aspartate-α-decarboxylase is responsible for decarboxylating L-aspartate to produce β-alanine in the biosynthetic pathway. Wild-type L-aspartate-α-decarboxylase has problems such as low activity, poor thermal stability and mechanism-induced inactivation. Although a large number of targeted transformation results have been reported, the improvement is not large, and L-aspartate is used as a catalytic substrate, which is costly. Summary of the invention

[0004] In view of this, the present invention provides an aspartate ammonia lyase mutant and its application. The mutant is LmAspA-Mut, which has the ability to efficiently catalyze the addition of ammonia to high-concentration acrylic acid to produce β-alanine

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] An aspartate ammonia lyase mutant is obtained by causing at least one mutation of T189V, M323I, K326M and N328A in a wild-type aspartate ammonia lyase.

[0007] The present invention selects aspartate ammonia lyase LmAspA derived from Lysinibacillus manganicus as the research object, and successfully obtains a mutant containing four-site mutations through homology modeling, molecular docking and introduction of mutations. The four-site mutations include at least one of T189V, M323I, K326M and N328A.

[0008] Among them, the mutant with the above four mutations is recorded as LmAspA-Mut, and its amino acid sequence is shown in SEQ ID NO.4.

[0009] Aspartate ammonia lyase LmAspA does not have the activity of catalyzing the addition of ammonia to acrylic acid, while the aspartate ammonia lyase mutant LmAspA-Mut obtained by the modification of the present invention exhibits extremely high activity in catalyzing the addition of ammonia to acrylic acid to generate β-alanine, and is a biocatalyst with great application potential.

[0010] In the present invention, the amino acid sequence of the wild-type aspartate ammonia-lyase is shown in SEQ ID NO.1.

[0011] The present invention also provides a nucleic acid encoding the aspartate ammonia lyase mutant.

[0012] In some embodiments, the nucleotide sequence of the nucleic acid encoding the aspartate ammonia-lyase mutant is shown in SEQ ID NO.5.

[0013] The present invention also provides a biological material comprising the nucleic acid, wherein the biological material is an expression vector or a recombinant host.

[0014] 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:

[0015] The aspartate ammonia lyase LmAspA gene shown in SEQ ID NO.2 was codon optimized to obtain an optimized LmAspA gene, the nucleotide sequence of which is shown in SEQ ID NO.3, and the amino acid sequence of the corresponding aspartate ammonia lyase 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 a recombinant plasmid pET28a-LmAspA. The recombinant plasmid pET28a-LmAspA is used as a template, and the whole plasmid is amplified by reverse PCR using primers with mutant bases. The obtained PCR product is digested with the methylated template by DpnⅠ enzyme, and the enzyme digestion product is transformed into Escherichia coli E.coli BL21 (DE3), so as to obtain the engineered bacteria E.coli BL21 (DE3) / pET28a-LmAspA-Mut containing the aspartate ammonia lyase mutant LmAspA-Mut gene, wherein the expression vector containing the recombinant aspartate ammonia lyase mutant encoding gene is named pET28a-LmAspA-Mut.

[0016] The present invention also provides the use of the aspartate ammonia lyase mutant, the nucleic acid, or the biological material in the preparation of β-alanine.

[0017] Specifically, in the above application, the aspartate ammonia lyase mutant, the nucleic acid, or the biological material is used to catalyze the addition of ammonia to acrylic acid to prepare β-alanine.

[0018] The present invention also provides a method for preparing β-alanine, comprising:

[0019] Fermenting and culturing a recombinant host expressing the aspartate ammonia lyase mutant of the present invention to obtain wet cells;

[0020] The wet bacteria 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 liquid is separated and purified to obtain β-alanine.

[0021] In some embodiments, the method for preparing wet bacteria comprises: preparing seed solution using a recombinant host expressing the aspartate ammonia lyase mutant of the present invention, then culturing and inducing expression to obtain induced culture solution, then centrifuging the induced culture solution, discarding the supernatant, and collecting wet bacteria.

[0022] In some specific embodiments, the method for preparing the wet cells comprises:

[0023] The engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut containing the gene encoding the aspartate ammonia lyase mutant were inoculated into LB liquid medium containing 100 μg / mL kanamycin, and cultured at 37 ° C for 12 h to obtain seed liquid. The seed liquid was inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37 ° C until OD 600 The pH value was 0.5-0.7, and then IPTG was added with a final concentration of 0.2 mM. The culture was induced at 24°C for 10 h to obtain the induced culture solution. The induced culture solution was then centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the wet bacteria were collected.

[0024] In the method for preparing β-alanine provided by the present invention, the reaction is: reacting for 5 hours at 25-60°C and 50-400rpm; in some specific embodiments, the reaction is: reacting for 5 hours at 40°C and 150rpm. In some specific embodiments, in the reaction system, the amount of catalyst used is 40g / L based on wet bacteria, the amount of substrate acrylic acid added is 315g / L, and the amount of ammonia water added is 125g / L.

[0025] In the present invention, the separation and purification of the reaction solution includes: separating and purifying the product by organic solvent precipitation method; in some specific embodiments, the separation and purification of the reaction solution includes: separating and purifying the product by organic solvent precipitation method; 无水乙醇 :V 反应液上清 Add ethanol in a ratio of 8:1, let it stand at room temperature for 2 hours, filter out the supernatant, and dry the crystallized product at 60°C to obtain β-alanine.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that: aspartate ammonia lyase LmAspA does not have the ability to catalyze the addition of ammonia of acrylic acid to generate β-alanine, and by introducing four-point mutations T189V / M323I / K326M / N328A, the aspartate ammonia lyase mutant LmAspA-Mut is endowed with the ability to efficiently catalyze the addition of ammonia of high-concentration acrylic acid to generate β-alanine. The wet bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut expressing the mutant LmAspA-Mut is used as a biocatalyst, 315 g / L acrylic acid and 125 g / L ammonia water are used as substrates to construct a reaction system, and the reaction is carried out at 40°C and 150 rpm for 5 hours, and the yield of β-alanine is as high as 99%, and the product concentration is 385.5 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of aspartate ammonia lyase catalyzing the addition of alanine to synthesize β-alanine;

[0028] Figure 2It is the agarose gel electrophoresis diagram of aspartate ammonia lyase encoding gene LmAspA and pET-28a; Lane M is a marker; Lane 1 is aspartate ammonia lyase encoding gene LmAspA; Lane 2 is pET-28a;

[0029] Figure 3 The agarose gel electrophoresis diagram of plasmid pET28a-LmAspA after inverse PCR amplification; Lane M is a marker; Lanes 1, 2, and 3 are plasmid inverse PCR products;

[0030] Figure 4 The figure is the SDS-PAGE image of the aspartate ammonia lyase mutant LmAspA-Mut; Lane M is a marker; Lane 1 is the crude enzyme solution prepared from the wet bacteria E.coli BL21(DE3) / pET28a-LmAspA-Mut without induction; Lane 2 is the crude enzyme solution prepared from the wet bacteria E.coli BL21(DE3) / pET28a-LmAspA-Mut after induction; Lane 3 is the pure enzyme solution obtained by separation and purification of the aspartate ammonia lyase mutant LmAspA-Mut;

[0031] Figure 5 This is the HPLC detection spectrum of β-alanine;

[0032] Figure 6 It is the HPLC detection standard curve of β-alanine;

[0033] Figure 7 The process of acrylic acid ammonia addition catalyzed by aspartate ammonia lyase mutant LmAspA-Mut;

[0034] Figure 8 This is the HPLC detection spectrum of β-alanine standard with a purity of 99%;

[0035] Fig. 9 The HPLC detection spectrum of β-alanine separated and purified from the reaction solution;

[0036] Fig.10 For β-alanine standard 1 H NMR(a) and 13 C NMR (b) spectrum;

[0037] Fig.11 To separate and purify the obtained β-alanine from the reaction solution 1 H NMR(a) and 13 C NMR (b) spectrum. DETAILED DESCRIPTION

[0038] The present invention provides an aspartate ammonia lyase mutant and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point 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 methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

[0040] The present invention will be further described below in conjunction with embodiments:

[0041] Example 1: Construction of genetically engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA

[0042] The aspartate ammonia lyase LmAspA encoding gene from Lysinibacillus manganicus (nucleotide sequence as shown in SEQ ID NO.2) was codon optimized, and the nucleotide sequence of the codon-optimized LmAspA gene was shown in SEQ ID NO.3, and the corresponding amino acid sequence of aspartate ammonia lyase LmAspA was shown in SEQ ID NO.1, which was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The codon-optimized LmAspA encoding gene (SEQ ID NO.3) was artificially synthesized and inserted between EcoRⅠ and HindⅢ of pET28a to obtain the recombinant plasmid pET28a-LmAspA. The agarose gel electrophoresis of the aspartate ammonia lyase encoding gene LmAspA and pET-28a is shown in Figure 2 shown.

[0043] Take out 5 μL of the synthesized recombinant expression plasmid pET28a-LmAspA and add it to 50 μL of E.coli BL21 (DE3) competent medium, flick the tube wall to mix, and place on ice for 30 minutes. Heat shock in a 42℃ water bath for 90 seconds, and immediately place on ice for 5 minutes. Add 900 μL of LB liquid culture medium to the tube and culture on a shaker at 37℃ for 1 hour. Centrifuge the culture solution at 4000rpm for 3 minutes and take out 900 μL of supernatant. Use the remaining culture medium to suspend the bacteria, take out 100 μL and apply it on LB solid culture medium containing 100 μg / mL kanamycin. Culture overnight in a 37℃ incubator for 14 hours to obtain the genetically engineered bacteria E.coli BL21 (DE3) / pET28a-LmAspA.

[0044] LB liquid culture medium composition: yeast extract 5 g / L, trypsin 10 g / L, NaCl 10 g / L, solvent is distilled water, pH 7.0-7.5.

[0045] Example 2: Construction of recombinant expression plasmid pET28a-LmAspA-Mut and genetically engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut

[0046] 1. Recombinant expression plasmid pET28a-LmAspA-Mut

[0047] The plasmid pET28a-LmAspA prepared in Example 1 was used as a template to design primers, and single-point mutation, three-point combination mutation, and four-point combination mutation of key amino acids (T189 / M323 / K326 / N328) were performed to find the best combination. The whole plasmid was cloned using inverse PCR technology and transformed into Escherichia coli E.coli BL21 (DE3). The plasmid was extracted for sequencing, and the sequencing results were analyzed using software. The sequence contained an open reading frame of 1410 bp in length, and a mutant plasmid was obtained in which the threonine at position 189 was successfully changed to valine, the methionine at position 323 was mutated to isoleucine, the lysine at position 326 was mutated to methionine, and the asparagine at position 328 was mutated to alanine. The mutant plasmid was then transformed into E. coli BL21 (DE3), and the plasmid was extracted and sequenced to successfully obtain the mutant plasmid pET28a-LmAspA-Mut. The nucleotide sequence of the mutant LmAspA-Mut is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.4. The primers are as follows:

[0048] M1-F 5'-GGTCGCACTCATCTGCAGGATGCAGTTCCG-3' (SEQ ID NO. 6);

[0049] M1-R 5'-CAGATGAGTGCGACCCATTTTAATGATGCC-3' (SEQ ID NO. 7);

[0050] M2-F 5'-AGCATTATGCCGGGTATGGTTGCACCGGTT-3' (SEQ ID NO. 8);

[0051] M2-R 5'-ACCCGGCATAATGCTGCTGCCAGGCTGACG-3' (SEQ ID NO.9);

[0052] M3-F 5’-CCGGGTAAGGTTGCACCGGTTATGCCGGAA-3’(SEQ ID NO.10);

[0053] M3-R 5’-TGCAACCTTACCCGGCATAATGCTGCTGCC-3’(SEQ ID NO.11);

[0054] M4-F 5’-AAGGTTAATCCGGTTATGCCGGAAGTTATG-3’(SEQ ID NO.12);

[0055] M4-R 5’-AACCGGATTAACCTTACCCGGCATAATGCT-3’(SEQ ID NO.13);

[0056] M5-F 5’-GGACGTACACATCTTCAAGATGCTGTTCCA-3’(SEQ ID NO.14);

[0057] M5-R 5’-AAGATGTGTACGTCCCATTTTAATAATGCC-3’(SEQ ID NO.15);

[0058] M6-F 5’-TCAATTATGCCAGGTATGGTGGCCCCTGTT-3’(SEQ ID NO.16);

[0059] M6-R 5’-ACCTGGCATAATTGATGAACCAGGCTGTCT-3’(SEQ ID NO.17);

[0060] M7-F 5’-CCAGGTAAAGTGGCCCCTGTTATGCCAGAA-3’(SEQ ID NO.18);

[0061] M7-R 5’-GGCCACTTTACCTGGCATAATTGATGAACC-3’(SEQ ID NO.19);

[0062] M8-F 5’-AAAGTGAACCCTGTTATGCCAGAAGTTATG-3’(SEQ ID NO.20);

[0063] M8-R 5’-AACAGGGTTCACTTTACCTGGCATAATTGA-3’(SEQ ID NO.21);

[0064] M9-F 5'-GGACGTACACATCTTCAAGATGCTGTTCCA-3' (SEQ ID NO. 22);

[0065] M9-R 5'-AAGATGTGTACGTCCCCATTTTAATAATGCC-3' (SEQ ID NO. 23).

[0066] The inverse PCR amplification system is shown in Table 1.

[0067] Table 1 PCR amplification reaction system

[0068]

[0069] 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. The denaturation and extension steps were repeated 30 times, and the temperature was lowered to 4°C after extension at 72°C for 10 min. The PCR products were detected by 0.8% agarose gel electrophoresis. Figure 3 A bright band can be seen at about 6500bp, which is consistent with the theoretical value of the plasmid.

[0070] 2. Genetically engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut

[0071] The PCR product was digested at 37°C for 1 h to remove the methylated template. The enzyme digestion system is shown in Table 2.

[0072] Table 2 Digestion system of methylated templates in PCR products

[0073]

[0074] 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) was directly transformed into the expression host bacterium E. coli BL21 (DE3) to obtain the genetically engineered E. coli BL21 (DE3) / pET28a-LmAspA-Mut. After the transformant was verified by colony PCR, it was inoculated into LB liquid culture medium containing 100 μg / mL kanamycin, cultured at 37°C for 12 hours, and the bacteria were collected by centrifugation, the plasmid was extracted, and sent for sequencing. The sequencing results were analyzed by software. The amino acid sequence showed that the threonine at position 189 was successfully changed to valine, the methionine at position 323 was mutated to isoleucine, the lysine at position 326 was mutated to methionine, and the asparagine at position 328 was mutated to alanine.

[0075] Example 3: Inducible expression and purification of aspartate ammonia lyase LmAspA and its mutant LmAspA-Mut

[0076] Wet cells were obtained by shaking flask culture: the engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut containing the aspartate ammonia lyase mutant encoding gene was inoculated into LB liquid medium containing 100 μg / mL kanamycin, and cultured at 37 ° C for 12 h to obtain seed liquid, and the seed liquid was inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% inoculation, and cultured at 37 ° C until OD 600 The pH value was 0.5-0.7, and then IPTG was added with a final concentration of 0.2 mM. The culture was induced at 24°C for 10 h to obtain the induced culture solution. The induced culture solution was then centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the wet bacteria were collected.

[0077] Fermentation tank culture to obtain wet bacteria: The genetically engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut constructed in Example 2 was inoculated into LB liquid culture medium containing 100 μg / mL kanamycin, and cultured at 37 ° C for 12 h to obtain seed liquid. Operate in a clean bench and add 3 mL of kana resistance to MgSO 4 solution, ignite the inoculation loop, and place MgSO 4 / Kana solution was poured into the culture medium. The final concentration of kanamycin in the fermentation medium was 100 μg / mL. The seed solution was inoculated into the fermentation tank from the fire ring at a 3% inoculation rate. The speed was adjusted to 600 rpm, the ventilation volume was increased to 6 L / min, and cultured at 37°C until its OD 600 After 4-6 (about 3.5h), add IPTG to 50mL of LB liquid culture medium in a clean bench, and add the LB culture medium containing IPTG into the fermenter through the inoculation port under the protection of the fire ring. The final concentration of IPTG in the fermentation medium is 0.2mM. Reduce the temperature of the fermenter to 24°C through the circulating water system, adjust the speed to 500rpm, and continue to culture for 10h to induce the bacteria to express protein. Pour the fermentation broth into a centrifuge cup, centrifuge it at 4°C, 8000rpm for 10min with a high-speed refrigerated centrifuge, discard the supernatant, and resuspend the obtained bacteria with 50mM Tris-HCl buffer (pH 8.0) to wash the bacteria. Put the bacterial suspension into a 50mL centrifuge tube, centrifuge it at 4°C, 12000rpm for 10min, discard the supernatant, and collect the wet bacteria.

[0078] The wet cells were added with an appropriate amount of Tris-HCl (pH 8.0) buffer at a ratio of 1 g wet cells to 20 mL Tris-HCl buffer (pH 8.0), and ultrasonically disrupted at 40% power for 15 min (working for 1 s, resting for 3 s). The disrupted liquid was centrifuged at 4°C and 8000 rpm for 10 min, and the centrifugation was repeated three times to obtain the supernatant crude enzyme solution. According to Ni-NTA metal chelate affinity chromatography (purchased from Bio-Rad, referred to as Ni 2+ Column, column inner diameter 1.6cm, column height 15cm) instructions, take 15mL of crude enzyme solution and load it onto the pre-equilibrated Ni 2+ The impurities and target protein were eluted in the column in sequence with eluents containing 5 mM imidazole, 50 mM imidazole, 100 mM imidazole, 200 mM imidazole and 500 mM imidazole (the eluent composition: corresponding concentration of imidazole, 300 mM sodium chloride, the solvent is 50 mM Tris-HCl buffer, pH 8.0), the elution rate was 2.5 mL / min, and 3 column volumes were eluted with each concentration of eluent. The effluent corresponding to the eluent containing 200 mM imidazole was collected and desalted and concentrated by centrifugation at 4 ° C and 5000 rpm for 30 min with an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate, i.e., LmAspA-Mut (T189V / M323I / K326M / N328A) enzyme solution, was taken and stored at -20 ° C for use.

[0079] The purity of the pure enzyme solution of the aspartate ammonia lyase mutant LmAspA-Mut was verified by SDS-PAGE gel electrophoresis. The results of SDS-PAGE electrophoresis are as follows: Figure 4 As shown. The aspartate ammonia lyase mutant LmAspA-Mut is a single band after SDS-PAGE electrophoresis. The theoretical subunit size of the aspartate ammonia lyase mutant LmAspA-Mut is about 52 kDa, and its apparent size on SDS-PAGE electrophoresis is consistent with the theoretical molecular weight.

[0080] Similarly, according to the operation process of Example 3, wet cells of E. coli BL21 (DE3) / pET28a-LmAspA induced to express LmAspA can be obtained and used as a control for the whole cell catalytic reaction.

[0081] Example 4: HPLC analysis of β-alanine in the process of enzymatically catalyzing the ammonia addition of acrylic acid to produce β-alanine

[0082] Preparation of mobile phase: Phase A: Weigh 2.05g of anhydrous sodium acetate, transfer 1.5mL of glacial acetic acid, add 1L of ultrapure water, stir and dissolve, and filter through a 0.22μm water filter membrane. Phase B: Accurately measure 1L of liquid-grade methanol, filter through a 0.45μm organic filter membrane. After mixing phases A and B at a ratio of 1:1 (v:v), degas in an ultrasonic cleaner for 30 minutes to obtain the mobile phase.

[0083] HPLC detection conditions: Welchrom column (250×4.6 / 5μm) column; mobile phase flow rate was 1mL / min; detection wavelength was 360nm; detection temperature was set to 40℃. According to the peak time of β-alanine standard samples with 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 ( Figure 5 The retention times of 2.713, 3.148, 3.293, 5.822, 6.589, and 9.668 min are all by-product peaks from the derivatization reaction.

[0084] Prepare 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 M β-alanine aqueous solutions, take 100 μL of each into a 1.5 mL EP tube, add 100 μL of Na 2 CO 3 / NaHCO 3 Buffer (pH 9.0), 100 μL 1% 2,4-dinitrofluorobenzene-acetonitrile solution, react in a 60°C metal bath in the dark for 1 h, then add 700 μL NaH 2 PO 4 / Na 2 HPO 4 Buffer (pH 7.0). Centrifuge at 12000rpm for 5min, filter the supernatant with a 0.22μm filter membrane into a brown liquid phase bottle, and use HPLC to determine the β-alanine peak time and the peak area corresponding to each concentration. Plot the β-alanine concentration and the corresponding peak area to obtain a standard curve of β-alanine concentration and peak area, as shown in Figure 6 The standard curve equation is y = 7.0407 × 10 8 X+3.3855×10 6 , y is the peak area, x is the β-alanine concentration (in M), R 2 The linear relationship is good.

[0085] Centrifuge the catalytic reaction solution at 12000rpm for 10min. Take 100μL of the supernatant and add 900μL of ultrapure water. Repeat once for a total dilution of 100 times. Take 100μL of the diluted reaction solution in a 1.5mL EP tube and add 100μL of Na 2 CO 3 / NaHCO 3 Buffer (pH 9.0), 100 μL 1% 2,4-dinitrofluorobenzene-acetonitrile solution, react in a 60°C metal bath in the dark for 1 h, then add 700 μL NaH 2 PO 4 / Na 2 HPO 4 Buffer (pH 7.0). Centrifuge at 12000rpm for 5min, take the supernatant and filter it with a 0.22μm filter membrane into a brown liquid phase bottle, and detect the peak area of ​​β-alanine corresponding to the catalytic reaction liquid of each mutant by HPLC, so as to compare the catalytic ability.

[0086] Example 5: Preparation of β-alanine by adding ammonia to acrylic acid catalyzed by the aspartate ammonia lyase mutant LmAspA-Mut

[0087] The wet bacteria obtained by fermentation culture of the engineered bacteria E. coli BL21 (DE3) / pET28a-LmAspA-Mut containing the gene encoding the aspartate ammonia lyase mutant were used as catalysts. The catalyst dosage was 40 g / L. The substrates 315 g / L acrylic acid and 125 g / L ammonia water were added, and water was added to make up the reaction system to 10 mL. The constructed 10 mL catalytic reaction system was reacted in a water bath shaker at 40 ° C and 150 rpm. 10 μL of the reaction solution was diluted 100 times every 1 h. After the DNFB derivatization reaction, the β-alanine content was detected by HPLC. The β-alanine concentration was calculated according to the standard curve, and the product yield was further calculated. The relationship between the product yield and the reaction time is shown in the figure. Figure 7 As shown in the figure, in a 10mL reaction system, when the reaction temperature is 40℃, the yield of β-alanine can reach 99% in 5h, and the corresponding product concentration is 385.5g / L. Under the same conditions, the wet bacteria obtained by fermentation culture of the engineered bacteria E.coli BL21(DE3) / pET28a-LmAspA containing the aspartate ammonia lyase encoding gene was used as a control, and no β-alanine was detected in the reaction solution, indicating that it is the introduced mutations T189V / M323I / K326M / N328A that endows the aspartate ammonia lyase mutant LmAspA-Mut with the ability to efficiently catalyze the addition of ammonia to high-concentration acrylic acid to generate β-alanine.

[0088] The reaction solution was centrifuged and the supernatant was collected. 乙醇 :V 反应上清液Anhydrous ethanol was added to the supernatant at a ratio of 8:1, and the mixture was allowed to stand at room temperature for 2 hours. The product precipitated as prismatic crystals. After the supernatant was filtered and discarded, the crystal product was dried at 60°C to obtain β-alanine. 3.56 mg of 99% pure β-alanine standard sample and 3 mg of purified β-alanine powder were dissolved in 1 mL of ultrapure water, and 100 μL of each was taken for DNFB derivatization reaction and then HPLC detection. The detection spectrum and results are shown in FIG. Figure 8 and Fig. 9 The retention time of β-alanine standard is 6.158min, and the corresponding peak area is 33744935 ( Figure 8 ), the retention time of the purified β-alanine was 6.166 min, and the corresponding peak area was 28198334 ( Fig. 9 ), the purity of β-alanine was calculated to be 98.17%.

[0089] Dissolve 10 mg of 99% pure β-alanine standard sample and purified β-alanine powder in 700 μL of 2 O, and NMR detection was performed. The detection spectrum is shown in Fig.10 and Fig.11 , the purified product and β-alanine standard 1 H NMR spectra and 13 The C NMR spectra were consistent, and the product was confirmed to be β-alanine.

[0090] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An aspartate ammonia lyase mutant, the amino acid sequence of which is shown in SEQ ID NO.

4.

2. A nucleic acid encoding the aspartate ammonia-lyase mutant according to claim 1.

3. The nucleic acid according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

5.

4. A biological material comprising the nucleic acid according to claim 2 or 3, wherein the biological material is an expression vector or a recombinant host.

5. Use of the aspartate ammonia-lyase mutant according to claim 1, the nucleic acid according to claim 2 or 3, or the biomaterial according to claim 4 in the preparation of β-alanine.

6. The use according to claim 5, characterized in that: The application is: catalyzing the addition of ammonia to acrylic acid to prepare β-alanine.

7. A method for preparing β-alanine, characterized in that: include: Fermenting and culturing a recombinant host expressing the aspartate ammonia lyase mutant according to claim 1 to obtain wet cells; The wet bacteria or the aspartate ammonia lyase mutant according to claim 1 is used to catalyze the reaction of acrylic acid and ammonia water, and the reaction liquid is separated and purified to obtain β-alanine.

8. The method according to claim 7, characterized in that The method for preparing wet bacterial cells comprises: preparing seed liquid using a recombinant host expressing the aspartate ammonia lyase mutant of claim 1, then culturing and inducing expression to obtain an induced culture liquid, then centrifuging the induced culture liquid, discarding the supernatant, and collecting wet bacterial cells.

9. The method according to claim 7, characterized in that: The reaction is carried out at 40° C. and 150 rpm for 5 hours.

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