Aspartase mutant and its application

Abstract

The invention discloses an aspartase mutant, which has an activity of 9.5 times higher than that of the original sequence in producing β-alanine using acrylic acid as a substrate, and can be used for industrial enzymatic catalysis to produce β-alanine. When the aspartase mutant whole cell catalyzes 150g / L acrylic acid substrate, the β-alanine production rate exceeds 97%.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme catalysis, and in particular relates to an aspartase mutant and its use in producing beta-alanine from acrylic acid by enzymatic catalysis. Background Art

[0002] β-Alanine is a natural β-amino acid that is not composed of proteins. It is a precursor compound for synthesizing muscle-derived active peptides such as carnosine and anserine. As a sports nutrition supplement for enhancing muscle endurance, it has been widely used in clinical nutrition. β-Alanine is also an important precursor side chain for the synthesis of calcium pantothenate (vitamin B5) in organisms, and is also an important raw material for the synthesis of calcium pantothenate in industry. As a functional amino acid, β-Alanine has been widely used in many industries such as medicine, food, and chemical industry.

[0003] The biological method for preparing β-alanine has become a development trend in recent years. The biological method mainly includes fermentation method and biocatalysis method. The fermentation method uses glucose as the starting raw material. The raw materials are cheap and easy to obtain, and has always been a research hotspot. For example, patent document CN112662609A discloses that Escherichia coli engineered bacteria uses glucose as the raw material to ferment 75.6g / L of β-alanine, and the sugar-acid conversion rate is 66.2%. In addition, patent documents CN112662609A, CN112625985A, and CN103898033A also disclose the use of Escherichia coli engineered bacteria to ferment to produce β-alanine. However, the fermentation method has many fermentation by-products, complex extraction process, high cost, etc., resulting in the fermentation method being in the laboratory research stage. There are two main biocatalytic processes: one is to use L-aspartate as a substrate, and L-aspartate α-decarboxylase (ADC) catalyzes decarboxylation to generate β-alanine. For example, the inventor's previous research document CN108546697A adopted this route. This method has high catalytic efficiency, mild conditions, simple and environmentally friendly extraction process, and has long been a hot spot in the study of enzymatic synthesis of β-alanine. However, this method requires the use of relatively expensive substrate aspartate, which leads to high process costs and great restrictions on industrialization; the other is to use acrylic acid as a substrate, and aspartase (also known as aspartate ammonia lyase (AspB), aspartate lyase) catalyzes the addition of ammonia, such as the document (Converting Aspartase into a b-Amino Acid Lyase by Cluster Screening. Andreas Vogel, et al., ChemCatChem 2014,6,p965-968.DOI:10.1002 / cctc.201300986), patent document CN109385415A, and the inventor's previous research document CN110791493A all adopted this route. This method was rapidly promoted to industrial development and application because the substrate is cheap and easy to obtain, the enzyme catalytic reaction and extraction process are relatively simple, and the production cycle is short. However, the process currently still has problems such as high production cost due to large enzyme dosage and high impurity content in the reaction process. Therefore, the process needs to be further improved in order to achieve better economic benefits. Summary of the invention

[0004] In order to improve the economic efficiency of aspartase-catalyzed acrylic acid ammonia reaction to prepare β-alanine, we focused our research on improving aspartase, including improving enzyme activity, substrate tolerance, and enzyme stability. With the development and application of bioinformatics technology, we have a certain understanding of the structure and active center of aspartase disclosed in the document DOI:10.1002 / cctc.201300986 and patent document CN110791493A, for example Figure 1The 3D model of the protein sequence of the initial aspartase (SEQ ID NO: 1) established by computer simulation technology is shown. Therefore, the aspartase (T187C, M321I, K324M, N326C) mutant (SEQ ID NO: 1) disclosed in the document DOI: 10.1002 / cctc.201300986 is further modified to improve its enzyme activity in catalyzing the ammonia addition reaction of acrylic acid, reduce impurities in the reaction process, thereby further reducing the production cost of β-alanine and improving product quality. Specifically, the present invention includes the following technical solutions:

[0005] An aspartase mutant is a mutant formed by mutation of one or more, preferably two or more, more preferably three or more, more preferably four or more of the following sites in the amino acid sequence of the initial aspartase SEQ ID NO: 1: R40, I41, H42, P43, E44, L45, I321, P322, G323, M324, V325, C326. The aspartase mutant has more than 98%, preferably more than 99% homology with the amino acid sequence of SEQ ID NO: 1, has the function of the aspartase SEQ ID NO: 1, preferably the function of catalyzing the ammonia addition reaction of acrylic acid to generate β-alanine, but has higher enzyme activity.

[0006] The above term "mutation" includes but is not limited to substitution, deletion or addition of amino acids.

[0007] Preferably, the aspartase mutant is a mutant formed by mutation of one or more of the following sites in the amino acid sequence of aspartase SEQ ID NO: 1: I41, H42, P43, C326. Preferably, sites I41, H42, P43 are mutated to aliphatic amino acids (G, A, V, L, I), respectively, and C326 is mutated to hydroxyl amino acids T or S.

[0008] Preferably, the above-mentioned aspartase mutant is a mutant formed by one or more mutations selected from the following group in the amino acid sequence of aspartase SEQ ID NO: 1: I41L, H42V, P43V, C326T.

[0009] More preferably, the above-mentioned aspartase mutant is an I41L, H42V, P43V, C326T mutant of SEQ ID NO: 1, and its amino acid sequence is SEQ ID NO: 3:

[0010] MNTDVRIEKDFLGEKEIPKDAYYGVQTIRATENFPITGYR LVVELIKSLGIVKKSAALANMEVGLLDKEVGQYIVKAADEVIEGKWNDQFIVDPIQGGAGTSINMNANEVIANRALELMGEEKGNYSKISPNSHVNMSQSTNDAFPTATH IAVLSLLNQLIETTKYMQQEFMKKADEFAGVIKMGRCHLQDAVPILLGQEFEAYARVIARDIERIANTRNNLYDINMGATAVGTGLNADPEYISIVTEHLAKFSGHP LRSAQHLVDATQNTDCYTEVSSALKVCMINMSKIANDLRLMASGPRAGLSEIVLPARQPGSSIIPGMVTPVMPEVMNQVAFQVFGNDLTITSASEAGQFELNVMEPV LFFNLIQSISIMTNVFKSFTENCLKGIKANEERMKEYVEKSIGIITAINPHVGYETAAKLAREAYLTGESIRELCIKYGVLTEEQLNEILNPYEMTHPGIAGRK(SEQ ID NO:3).

[0011] The second aspect of the present invention provides a gene encoding the above-mentioned aspartase mutant.

[0012] Preferably, the nucleotide sequence of the gene encoding the aspartase mutant SEQ ID NO:3 may be SEQ ID NO:4.

[0013] The third aspect of the present invention provides a plasmid comprising the above gene. The plasmid comprises a vector for expressing the above gene, preferably a vector of the PET series, such as pET22b, pET24a, pET28a, etc., but is not limited thereto.

[0014] The fourth aspect of the present invention provides a microorganism for expressing the above-mentioned aspartase mutant. For example, the microorganism is a transformant transformed with the above-mentioned plasmid.

[0015] The above-mentioned microorganism can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0016] The fifth aspect of the present invention provides use of the above-mentioned aspartase mutant or the above-mentioned microorganism in producing β-alanine.

[0017] Specifically, in the production of β-alanine, acrylic acid or acrylonitrile, preferably acrylic acid, can be used as a substrate raw material, and the above-mentioned aspartase mutant or microorganism can be used as a catalyst to catalyze the reaction.

[0018] As an optional embodiment, the above-mentioned microorganism can be in the form of bacterial cells or cell fragments thereof, and used as a catalyst for the acylation reaction.

[0019] Conventional process conditions can be used to produce β-alanine. For example, the concentration of acrylic acid in the reaction system can be selected to be 2-20wt%, preferably 15wt%; the reaction temperature can be selected to be 30-65°C, preferably 40-60°C, more preferably 45-55°C, and most preferably 50±0.5°C.

[0020] An ammonia source is also added to the above reaction system, and the ammonia source can be aqueous ammonia or an inorganic ammonium salt (such as ammonium sulfate, ammonium carbonate or ammonium nitrate), preferably aqueous ammonia.

[0021] The pH of the reaction system may be 8.5-10.0, preferably pH 9.0-9.8, more preferably about pH 9.5.

[0022] In the above reaction system, the substrate concentration may be 50-150 g / L, ie, 5-15 wt%.

[0023] Compared with the initial aspartase SEQ ID NO: 1, the aspartase mutant SEQ ID NO: 3 constructed in the present invention has significantly improved enzyme activity in catalyzing acrylic acid reaction. When the aspartase mutant is used to produce β-alanine by enzymatic method, the β-alanine production rate can reach 97% at a substrate concentration of 150 g / L, thereby improving the feasibility of industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the 3D modeling diagram of the protein sequence of the initial aspartase SEQ ID NO: 1

[0025] Figure 2 The liquid phase comparison diagram of the mutant BL4-R4 strain catalyzing acrylic acid reaction at 0 h and 10 h is shown. DETAILED DESCRIPTION

[0026] The original aspartase SEQ ID NO: 1 as the object of modification of the present invention is also a mutant enzyme, and its wild type is derived from Bacillus sp. YM55-1 and has been described in the document DOI: 10.1002 / cctc.201300986.

[0027] Its amino acid sequence is:

[0028] MNTDVRIEKDFLGEKEIPKDAYYGVQTIRATENFPITGYRIHPELIKSLGIVKKSAALANMEVGLLDKEVGQYIVKAADEVIEGKWNDQFIVDPIQGGAGTSINMNANEVIANRALEL MGEEKGNYSKISPNSHVNMSQSTNDAFPTATHIAVLSLLNQLIETTKYMQQEFMKKADEFAGVIKMGRCHLQDAVPILLGQEFEAYARVIARDIERIANTRNNLYDINMGATAVGTGL NADPEYISIVTEHLAKFSGHPLRSAQHLVDATQNTDCYTEVSSALKVCMINMSKIANDLRLMASGPRAGLSEIVLPARQPGSSIIPGMVCPVMPEVMNQVAFQVFGNDLTITSASEAG QFELNVMEPVLFFNLIQSISIMTNVFKSFTENCLKGIKANEERMKEYVEKSIGIITAINPHVGYETAAKLAREAYLTGESIRELCIKYGVLTEEQLNEILNPYEMTHPGIAGRK(SEQ ID NO:1).

[0029] Based on the computer-simulated protein sequence 3D model and the computer simulation results of Wu Bian's laboratory at the Institute of Microbiology, Chinese Academy of Sciences in 2018 (Ruifeng Li.et al, Computational redesign of enzymes for regio-and enantioselective hydroamination.Nat.Chem.Biol.,2018), we speculated that the 40-45 region and the 321-326 region of aspartase SEQ ID NO:1 are closely related to the enzyme active center and substrate / reactant channel, and focused on the mutations in this region. Through rational analysis and semi-rational design, the initial aspartase was mutated to improve the enzyme activity.

[0030] In this article, the terms "initial (type)", "initial enzyme", and "initial type enzyme" have the same meaning, and all refer to aspartase with an amino acid sequence of SEQ ID NO: 1. For the sake of convenience, the initial type aspartase and its mutants may be collectively referred to as "aspartase" in this article.

[0031] The mutation of amino acids includes substitution, deletion or addition. Among them, the substitution of amino acids includes conservative substitution and non-conservative substitution. "Conservative substitution" refers to the interchangeability of residues with similar side chains, and therefore generally includes replacing the amino acids in the polypeptide with the same or similar amino acid definition category. For example, but not limited to, an amino acid with an aliphatic side chain can be replaced with another aliphatic amino acid such as alanine, valine, leucine and isoleucine; an amino acid with a hydroxyl side chain can be replaced with another amino acid with a hydroxyl side chain such as serine and threonine; an amino acid with an aromatic side chain can be replaced with another amino acid with an aromatic side chain such as phenylalanine, tyrosine, tryptophan and histidine; an amino acid with a basic side chain can be replaced with another amino acid with a basic side chain such as lysine and arginine; an amino acid with an acidic side chain can be replaced with another amino acid with an acidic side chain such as aspartic acid or glutamic acid; and a hydrophobic amino acid or a hydrophilic amino acid can be replaced with another hydrophobic amino acid or a hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided below:

[0032]

[0033] "Non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may utilize amino acids between defined groups rather than within them, and affect: (a) the structure of the peptide backbone in the region of the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0034] The mutations at some sites in the amino acid sequence of the initial enzyme are not single mutations, for example, the mutation at position 41 may be I41L, I41A or I41G, in which case it may be expressed as I41LAG. The I41L mutation refers to a mutation in which the residue isoleucine (I or Ile) at position 41 of the amino acid sequence of SEQ ID NO: 1 is replaced by leucine (L or Leu), and so on.

[0035] For the sake of convenience, the abbreviations of amino acids in proteins can be expressed in English using either three letters or single letters, which are well known to those skilled in the art. These abbreviations are listed in the following table:

[0036] Table 1 Chinese and English correspondence and abbreviations of amino acids

[0037]

[0038]

[0039] In order to obtain aspartase with higher enzyme performance, the present invention uses Escherichia coli as a host bacterium, and performs point mutation on the coding gene sequence SEQ ID NO:2 of the initial aspartase SEQ ID NO:1. By screening the double-site saturation mutation library at positions 324 and 326, a mutant enzyme 1 at the threonine position 326 is obtained, and then homology modeling is performed on the mutant enzyme 1, and on the basis of the amino acid sequence of the mutant enzyme 1, single-point saturation mutation or multi-point saturation mutation is further performed on the 41st, 42nd and 43rd sites that affect substrate binding, respectively, to obtain mutant enzyme 2 at the 41st leucine site, mutant enzyme 3 at the 42nd valine and 43rd valine sites, and then a mutant enzyme 4 with mutations at four sites relative to the initial enzyme SEQ ID NO:1 at positions 41, 42nd valine, 43rd valine and 326th threonine is obtained by combined mutation, and its amino acid sequence is SEQ ID NO:3.

[0040] The aspartase mutant of the present invention has only 468 amino acids and a clear structure, so those skilled in the art can easily obtain its encoding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmids.

[0041] These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

[0042] As is known to all, the expression results of the same nucleotide sequence in different microbial hosts are often very different. In order to optimally express aspartase or its mutants in Escherichia coli, which is the most commonly used in genetic engineering, the expression genes of these enzymes can be codon optimized.

[0043] Codon optimization is a kind of technology that can be used for maximizing protein expression in organism by increasing the translation efficiency of gene of interest.Different organisms usually show the special preference for one of some codons of the same amino acid being encoded due to mutation tendency and natural selection.For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its respective genome tRNA library.Therefore, in fast-growing microorganisms, the low-frequency codon of amino acid can be replaced with the codon for the same amino acid but with high frequency.Therefore, the expression of optimized DNA sequence is improved in fast-growing microorganisms.

[0044] To express aspartase in E. coli, the gene encoding the codon-optimized original aspartase SEQ ID NO: 1 may be SEQ ID NO: 2; the gene encoding the aspartase mutant SEQ ID NO: 3 may be SEQ ID NO: 4.

[0045] The above-mentioned transformant host can be any microorganism suitable for expressing aspartase, including bacteria and fungi. Preferably, the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0046] When used as a biocatalyst for producing β-alanine, the aspartase of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, enzymes immobilized on carriers, etc.; the bacterial cell forms include living bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0047] As another optional embodiment, a microbial cell expressing the above-mentioned aspartase can be used as a biocatalyst for an enzyme-catalyzed reaction. The microorganism can be in the form of a cell or a cell fragment thereof, and the cell form includes a living cell and a dead cell, because when a microorganism such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli is no longer fermented and proliferated, but is used for an enzyme-catalyzed reaction, it is itself a natural immobilized enzyme, and does not require a crushing process, or even an extraction and purification process, and can be used as an enzyme preparation for a catalytic reaction. Since both the reaction substrate and the reaction product are small molecule compounds, they can easily pass through the cell membrane, the biological barrier of the cell, and therefore do not need to be crushed, which is economically advantageous.

[0048] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0049] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0050] Example

[0051] Materials and methods

[0052] The whole gene synthesis, primer synthesis and sequencing in the examples were all commissioned to Suzhou Genewise Biotechnology Co., Ltd.

[0053] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0054] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0055] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0056] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4.3H2O, 2.31g / L KH2PO4, 5g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20g / L agar powder.)

[0057] HPLC determination conditions of substrate acrylic acid and product β-alanine:

[0058] Agilent 1260 high performance liquid chromatograph; HLIIC Silica 5μm column, 4.6×250mm; detection wavelength 210nm; mobile phase A: 40mM potassium dihydrogen phosphate; mobile phase B: acetonitrile; mobile phase A: mobile phase B = 30:70; operating temperature 30℃; flow rate 1ml / min;

[0059] It should be noted that, for the convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme encoding gene number may share one number, which is easy for those skilled in the art to understand, that is, the same number can refer to different biological forms in different environments. For example, BL4 can represent the initial aspartase expression strain, the plasmid pET24a-BL4 number, the initial enzyme SEQ ID NO:1 number, and the initial enzyme encoding gene SEQ ID NO:2 number.

[0060] Example 1: Construction of recombinant Escherichia coli with initial aspartase gene

[0061] The aspartase (T187C, M321I, K324M, N326C) mutant disclosed in the document DOI: 10.1002 / cctc.201300986 was used as the initial enzyme, and its amino acid sequence was SEQ ID NO: 1. Based on this, codon optimization was performed, and the coding gene sequence SEQ ID NO: 2 was synthesized by full gene synthesis, and restriction endonuclease sites Nde I and XhoI were designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-BL4. The recombinant plasmid pET24a-BL4 was transformed into the expression host Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli BL4 expressing the initial aspartase.

[0062] Example 2: First round of mutation: establishment and screening of a double-site saturation mutation library

[0063] 2.1 Establishment of double-site saturation mutation library at positions 324 and 326

[0064] The gene SEQ ID NO: 2 of the initial enzyme was used as a template and amplified with degenerate codon primers to obtain a saturation mutation library at the 324th and 326th positions.

[0065] Forward primer BL4-F: 5'-GTTCCTCCATCATCCCGGGT NNK GTT NNK CCGGTTATGCCGGAAGTTATG-3',

[0066] Reverse primer BL4-R: 5'-CATAACTTCCGGCATAACCGG MNN AAC MNN ACCCGGGATGATGGAGGAAC-3',

[0067] Where M=A / C, K=G / T, N=A / G / C / T.

[0068] The 50 μL PCR reaction system includes: 10 ng plasmid template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0069] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0070] 1 μL DpnI was added to the amplified PCR product for digestion for 1 hour, and then electroporated into E. coli BL21 (DE3) competent cells to obtain more than 10 4 A random mutation library of 10 clones.

[0071] 2.2 High-throughput screening of mutant libraries

[0072] Transformants from the mutant library were selected and inoculated into 500 μL of LB liquid medium containing 50 μg / mL kanamycin in a 96-well deep-well culture plate, cultured overnight, and then 80 μL of the overnight culture was transferred to 800 μL of LB liquid medium containing 50 μg / mL kanamycin. After culturing at 37°C for 3 hours, IPTG was added at a final concentration of 0.5 mM, cooled to 25°C, and cultured overnight. Centrifuged at 4000 rpm for 15 minutes, discarded the supernatant, and 100 μL of sterile water was added to resuspend the cells for viability determination.

[0073] 2.3 High-throughput enzyme activity assay

[0074] Enzyme activity definition: The amount of enzyme required to catalyze the production of 1 micromole (μmol) of β-alanine from the substrate acrylic acid per minute at 45°C is defined as 1 unit (U).

[0075] Add 100 μL of the bacterial suspension in step 2.2 above to 100 μL of substrate reaction solution (10% acrylic acid, pH adjusted to 9.5 with concentrated ammonia, and 5 mM magnesium sulfate added), react at 50°C for 1-3 hours, and then centrifuge at 4°C, 5000 rpm for 10 minutes. Take the supernatant and measure the absorbance at 240 nm.

[0076] By screening about 3,000 mutant clones, the results showed that a clone BL4-R1 was obtained with a significantly enhanced ability to consume substrates. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to sequence the genome of strain BL4-R1, and by comparing the sequence with the initial enzyme, it was confirmed that the mutation site of the mutant was C326T.

[0077] Table 2. Relative activity of mutant bacteria

[0078] Strain No. Mutation site Relative specific activity of fermentation broth BL4 - 100％ BL4-R1 C326T 550％

[0079] The results showed that the C326T mutation was a positive mutation.

[0080] Example 3: Second round of mutation: establishment and screening of single-site saturation mutation point library

[0081] 3.1 Establishment of single-site saturation mutation library at position 41

[0082] Using the plasmid extracted from BL4-R1 as a template, a single-point saturation mutation library was established for the 41st site by amplification using degenerate codon primers.

[0083] The forward primer BL4-R1-F is:

[0084] 5'-CTTCCCGATCACCGGTTACCGT NNK CACCCGGAACTGATCAAATC-3',

[0085] The reverse primer BL4-R1-R is:

[0086] 5'-GATTTGATCAGTTCCGGGTG MNN ACGGTAACCGGTGATCGGGAAG-3',

[0087] Where M=A / C, K=G / T, N=A / G / C / T.

[0088] The 50 μL PCR reaction system includes: 10 ng plasmid template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0089] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0090] 1 μL DpnI was added to the amplified PCR product for digestion for 1 hour, and then electroporated into E. coli BL21 (DE3) competent cells to obtain more than 10 3 A random mutation library of 10 clones.

[0091] 3.2 High-throughput screening of mutant libraries

[0092] The method is the same as step 2.2 in Example 2.

[0093] 3.3 High-throughput enzyme activity assay

[0094] The method is the same as step 2.3 in Example 2. By screening about 200 mutant clones, the results show that a BL4-R2 strain with significantly enhanced ability to consume substrates was obtained. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to sequence the genome of strain BL4-R2, and it was determined that the 41st amino acid of aspartase expressed by the strain was mutated from isoleucine to leucine. The comparison results of bacterial activity are shown in Table 3 below.

[0095] Table 3. Relative activity of mutant bacteria in the second round

[0096] Strain No. Relative BL4 mutation site SEQ ID NO: Relative specific activity of fermentation broth BL4-R1 C326T 3 100％ BL4-R2 C326T、I41L 4 180％

[0097] The results showed that the I41L mutation was a positive mutation.

[0098] Example 4: The third round of mutation: establishment and screening of a double-site saturation mutation library

[0099] 4.1 Establishment of double-site saturation mutation library at positions 42 and 43

[0100] The plasmid extracted from BL4-R1 was used as a template and degenerate codon primers were used for amplification to obtain a saturation mutation library at both sites 42 and 43.

[0101] The forward primer BL4-R1-42&43-F is:

[0102] 5'-GATCACCGGTTACCGTATC NNKNNK GAACTGATCAAATCCCTG-3',

[0103] The reverse primers BL4-R1-42&43-R are:

[0104] 5'-CAGGGATTTGATCAGTTC MNNMNN GATACGGTAACCGGTGATC-3',

[0105] Where M=A / C, K=G / T, N=A / G / C / T.

[0106] The 50 μL PCR reaction system includes: 10 ng plasmid template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0107] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0108] 1 μL DpnI was added to the amplified PCR product for digestion for 1 hour, and then electroporated into E. coli BL21 (DE3) competent cells to obtain more than 10 4 A random mutation library of 10 clones.

[0109] 4.2 High-throughput screening of mutant libraries

[0110] The method is the same as step 2.2 in Example 2.

[0111] 4.3 High-throughput enzyme activity assay

[0112] The method was the same as step 2.3 in Example 2. About 3000 mutant clones were screened. The results showed that a clone BL4-R1 clone strain was obtained with a significantly improved ability to consume substrate. The 42nd amino acid of the strain was determined by sequencing to be mutated from histidine to valine, and the 43rd amino acid was mutated from proline to valine. The results of the cell activity comparison are shown in Table 4 below.

[0113] Table 4. Relative activity results of mutant bacteria

[0114] Strain No. Relative BL4 mutation site Relative specific activity of fermentation broth BL4-R1 C326T 100％ BL4-R3 C326T, H42V, P43V 140％

[0115] The results showed that H42V and P43V mutations were positive mutations.

[0116] Example 5: Fourth round of mutation: establishment of single-point directed mutation strain

[0117] 5.1 Construction of strains for single-site mutation at position 41

[0118] The plasmid extracted from the mutant strain BL4-R3 was used as a template and primer ring expansion was used to obtain the mutant strain at the 41st site.

[0119] The forward primer BL4-R3-41L-F is:

[0120] 5'-CTTCCCGATCACCGGTTACCGTTCTGGTGGTGGAACTGATCAAATC-3',

[0121] The reverse primer BL4-R3-41L-R is:

[0122] 5'-GATTTGATCAGTTCCACCACCAGACGGTAACCGGTGATCGGGAAG-3'.

[0123] The 50 μL PCR reaction system includes: 10 ng plasmid template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0124] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0125] The amplified PCR product was digested with 1 μL DpnI for 1 hour and then electroporated into competent E. coli BL21 (DE3).

[0126] 5.2 Enzyme activity determination

[0127] The method was the same as step 2.3 in Example 2. The results showed that the combined mutant strains of the three sites I41L, H42V, and P43V had higher enzyme activities than those of BL-R1, BL-R2, and BL-R3. The comparison results of bacterial activity measurements are shown in Table 5 below.

[0128] Table 5. Relative activity of mutant bacteria

[0129] Strain No. Relative BL4 mutation site Relative specific activity of fermentation broth BL4-R1 C326T 100％ BL4-R2 C326T、I41L 180％ BL4-R3 C326T, H42V, P43V 140％ BL4-R4 C326T, I41L, H42V, P43V 220％

[0130] The results showed that I41L mutation, H42V, and P43V mutations can be combined with C326T and have a positive additive effect, and their combination is a positive mutation.

[0131] Example 6: Strain fermentation comparison and transformation

[0132] 6.1 Shake flask fermentation

[0133] Single colonies were picked from the LB culture plates of the initial strain BL4 and the mutant strain BL4-R4, respectively, and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and cultured at 37°C, 250 rpm overnight. 2 mL of the overnight culture was inoculated into 200 mL of TB medium, and cultured at 37°C, 250 rpm for 2-3 hours until the OD 600 When the concentration was 0.6-0.8, 0.1 mM IPTG was added and cultured overnight at 28°C and 200 rpm. Then, the cells were collected by centrifugation at 4°C and 10,000 rpm for 10 min.

[0134] 6.2 Determination of bacterial specific activity

[0135] Reaction system: 10% acrylic acid, concentrated ammonia water to correct pH to 9.5, add magnesium sulfate with a final concentration of 5mM, 1% bacterial cells. React at 50°C for 30 minutes, take 500μl of the reaction solution, add 500μl of 2M HCl solution and mix well, centrifuge at 4°C, 12000rpm for 5min, take the supernatant, and determine the content of β-alanine by HPLC. The results are shown in Table 6.

[0136] Table 6. Results of enzyme activity assay of initial strain BL4 and mutant strain BL4-R4

[0137] Strain No. Mutation site Amino acid sequence number Relative Vitality BL4 —— 1 100％ BL4-R4 C326T, I41L, H42V, P43V 3 950％

[0138] As can be seen from Table 6, compared with the initial strain BL4 (expressing SEQ ID NO: 1), the enzyme activity of the aspartase mutant BL4-R4 (expressing mutant enzyme SEQ ID NO: 6) of the present invention is increased by 9.5 times.

[0139] Example 7: Test of β-alanine production by aspartase mutants

[0140] The pH of 15 wt% acrylic acid solution was adjusted to 9.5 with concentrated ammonia, and magnesium sulfate was added to a final concentration of 5 mM. Then, BL4-R4 frozen-thawed bacteria with a final concentration of 60,000 U / L enzyme activity were added and stirred at 50°C for reaction. The temperature was controlled at 50±1°C during the reaction and the reaction lasted for 16 hours. The reaction samples were tested by HPLC, see Figure 2The results showed that after 10 hours of reaction, the acrylic acid content in the reaction system was less than 0.1 g / L and the generation rate of β-alanine exceeded 97%.

[0141] In summary, compared with the original aspartase BL4, the aspartase mutant BL4-R4 (SEQ ID NO: 6) constructed in the present invention has significantly improved enzyme activity in catalyzing the reaction of acrylic acid to produce β-alanine, which is increased by 9.5 times, thereby enhancing the feasibility of industrial application.

Claims

1. An aspartase mutant, characterized in that: The amino acid sequence is SEQ ID NO:

3.

2. A gene encoding the aspartase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

4.

4. A plasmid for expressing the gene as claimed in claim 3.

5. A microorganism expressing the aspartase mutant according to claim 1.

6. The microorganism according to claim 5, characterized in that The microorganism is a transformant transformed with the plasmid according to claim 4.

7. The microorganism according to claim 6, characterized in that Selected from Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae.

8. The microorganism according to claim 7, characterized in that The microorganism is Escherichia coli BL21 (DE3).

9. Use of the aspartase mutant according to claim 1 or the microorganism according to claim 5 in producing β-alanine.

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