An aspartase variant, method of making and use thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses an aspartase variant, a preparation method and application thereof. The aspartase variant provided by the application has aspartase activity, has more than 98% homology with SEQ ID NO:1 in the amino acid sequence, and has I103F / T187I / M321I / K324I / N326C mutations; or further has D20V, I41L, H42V, P43V, N142V, L156F, H188A, L258I, A422T and / or Y429 mutations. The aspartase variant obtained by a large number of research screenings of the application has greatly improved enzyme activity under the same conditions relative to wild-type aspartase, is at least improved by more than 28 times, and can be up to 81 times, so that the conversion rate of acrylic acid in the acrylic acid ammonia reaction can be improved, and the aspartase variant has more practical value in the preparation of beta-alanine and derivatives thereof.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an aspartic acid enzyme variant, its preparation method, and its application. Background Technology

[0002] Aspartase (E.C.4.3.1.1), also known as aspartate ammonia-lyase, is widely found in bacteria, yeast, and plants. Aspartase derived from *E. coli* consists of four identical subunits. Active aspartases exist in tetrameric form. Aspartases belong to the fumarate superfamily, which includes aspartases, fumarates, and argininosuccinate lyases from various biological species. Aspartase-fumarate formic acid members do not require coenzymes or prosthetic groups for catalytic reactions; they are likely derived from a non-regulatory tetrasubunit enzyme that recognizes fumarate. All members of this family retain the common function of opening the fumarate double bond. Fumarate lyase catalyzes the opening of the fumarate double bond by water, argininosuccinate lyase catalyzes the opening of the fumarate double bond by arginine, and aspartase catalyzes the opening of the fumarate double bond by NH4+. 4+ It opens the double bond. Due to this specific function of aspartic acidase, it is widely used in industry to catalyze the production of L-aspartic acid from fumaric acid and ammonia, and this reaction is reversible.

[0003] Numerous studies have been conducted on the catalytic sites and reaction mechanisms of aspartic acid catalyzing the formation of L-aspartate from fumarate and ammonia by aspartase. Therefore, methods such as related mutations are employed to obtain aspartases with improved activity. For example, Chinese Patent 201710659654.9 discloses an aspartase variant with mutations at positions 187 and 326, namely T187I and N326C, and further variants such as D20V, V75E, Q89H, L156F, T164I, Y204C, N226I, L258I, M285L, M321I, K324I, K381R, K389I, I406L, R426C, and / or P456L. Compared to the wild-type parental aspartate enzyme, it does exhibit improved catalytic activity for the ammoniaation of acrylic acid, with superior thermal stability and pH profile, significantly increasing the conversion rate of acrylic acid in the ammoniaation reaction. For example, the aspartate enzyme mutant disclosed in Chinese Patent 202310796436.5 contains mutations at sites I41L, H42V, P43V, T187C, M321I, K324M, N326T, and A422T on the amino acid sequence of the wild-type aspartate enzyme. This mutant exhibits high temperature tolerance, thus shortening the reaction time for the production of β-alanine, and the system is less prone to bacterial contamination under these reaction conditions. However, obtaining an aspartate enzyme mutant with significantly improved activity for catalyzing the ammoniaation of acrylic acid to produce β-alanine remains a challenging task. Summary of the Invention

[0004] This invention builds upon previous research on mutations of aspartate enzymes and further explores related avenous compounds. Extensive research has yielded a series of aspartate enzyme variants with improved catalytic activity for the ammonia addition of acrylic acid, which can be used to catalyze the production of β-alanine.

[0005] This invention provides an aspartate enzyme variant with aspartate enzyme activity. Its amino acid sequence has more than 98% homology with SEQ ID NO: 1, and it contains the I103F / T187I / M321I / K324I / N326C mutation based on SEQ ID NO: 1. Or, based on this, at least one of the following mutations may also exist: D20V, I41L, H42V, P43V, N142V, L156F, H188A, L258I, A422T and / or Y429D.

[0006] Specifically, in addition to the I103F / T187I / M321I / K324I / N326C mutations compared to SEQ ID NO: 1, any of the following mutations also exist: D20V; I41L; H42V; P43V; D20V / L156F; D20V / A422T; D20V / Y429D; D20V / I41L / L156F; D20V / I41L / H188A; D20V / I41L / A422T; D20V / I41L / Y429D; D20V / H42V / P43V; D20V / H42V / N142V; D20V / N142V / L156F; D20V / L156F / H188A; D20V / L156F / A422T; D20V / H188A / L258I; D20V / L258I / A422T; D20V / A422T / Y429D; D20V / I41L / H42V / L156F; D20V / I41L / N142V / H188A; D20V / I41L / L258I / A422T; D20V / I42L / L156F / H188A; D20V / P43V / L156F / Y429D; D20V / P43V / A422T / Y429D; D20V / N142V / L156F / L258I; D20V / N142V / H188A / L258I; D20V / N142V / A422T / Y429D; D20V / L156F / H188A / A422T; D20V / L156F / A422T / Y429D; D20V / I41L / H42V / P43V / H188A; D20V / I41L / H42V / L156F / L258I; D20V / H42V / N142V / L156F / A422T; D20V / P43V / N142V / H188A / L258I; D20V / P43V / H188A / L258I / A422T; N142V; L156F; H188A; L258I; H42V / N142V; P43V / L156F; N142V / L258I; I41L / H42V / L156F; I41L / H42V / H188A; I41L / P43V / N142V; I41L / P43V / Y429D; I41L / N142V / A422T; I41L / N142V / H188A; I41L / L156F / L258I; I41L / L156F / Y429D; I41L / L258I / A422T; H42V / P43V / Y429D; H42V / N142V / L156F; H42V / L156F / H188A; H42V / L258I / A422T; I41L / H42V / P43V / Y429D; I41L / H42V / P43V / L258I; I41L / H42V / N142V / Y429D; I41L / H42V / H188A / A422T; I41L / N142V / L156F / L258I; I41L / H188A / L258I / Y429D; H42V / P43V / L156F / A422T; H42V / N142V / L156F / H188A; H42V / N142V / L258I / A422T; H42V / L156F / L258I / Y429D; H42V / H188A / L258I / A422T; D20V / N142V / L258I / A422T / Y429D; D20V / N142V / L156F / H188A / Y429D; I41L / H42V / P43V / N142V / L156F; I41L / H42V / P43V / H188A / A422T; I41L / P43V / N142V / L156F / L258I; A422T; Y429D; N142V / L156F; L258I / A422T; A422T / Y429D; P43V / N142V / L258I; P43V / L156F / Y429D; P43V / H188A / A422T; P43V / L258I / A422T; P43V / A422T / Y429D; N142V / L156F / L258I; L156F / H188A / L258I; L156F / H188A / Y429D; L156F / L258I / A422T; H188A / L258I / Y429D; H188A / A422T / Y429D; P43V / N142V / L156F / A422T; P43V / N142V / H188A / L258I; P43V / H188A / L258I / Y429D; N142V / L156F / H188A / A422T; N142V / L156F / A422T / Y429D; N142V / L156F / L258I / Y429D; N142V / H188A / A422T / Y429D; L156F / L258I / A422T / Y429D; L156F / H188A / L258I / A422T; I41L / N142V / H188A / A422T / Y429D; I41L / L156F / H188A / L258I / A422T; H42V / N142V / L156F / L258I / Y429D; P43V / N142V / H188A / L258I / A422T; N142V / L156F / L258I / A422T / Y429D; D20V / I41L / H42V / P43V / N142V / L258I; D20V / P43V / L156F / H188A / L258I / Y429D; I41L / H42V / N142V / L156F / L258I / A422T; I41L / P43V / L156F / L258I / A422T / Y429D; P43V / N142V / L156F / L258I / A422T / Y429D; I41L / H42V / L156F / H188A / L258I / A422T / Y429D.

[0007] The present invention also provides the nucleic acid encoding the aspartic acid enzyme variant. This nucleic acid can be DNA or RNA.

[0008] The present invention further provides an expression cassette, recombinant vector, transgenic cell line or recombinant cell containing the encoded nucleic acid.

[0009] The expression cassette typically contains a promoter that initiates transcription of the gene, and the gene and transcription termination sequence may also include, but are not limited to, a leader sequence, a polyadenylate sequence, a signal peptide sequence, or other regulatory expression elements.

[0010] The recombinant vector may be a recombinant cloning vector or a recombinant expression vector.

[0011] The host cell is a prokaryotic or eukaryotic cell. The prokaryotic host cell can be a Gram-positive or Gram-negative bacterial cell. Gram-positive bacteria include, for example, *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus marineensis*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. Gram-negative bacteria include, but are not limited to, *Escherichia coli*, *Campylobacter*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*.

[0012] The Bacillus genus cells mentioned include, for example, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus spheroidae, Bacillus circulosa, Bacillus croceae, Bacillus coagulans, Bacillus sclerosus, Bacillus brilliance, Bacillus sluggishness, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0013] The streptococcal cells include, for example, Streptococcus equi, Streptococcus pyogenes, and Streptococcus equi subsp. vesicanthioides cells.

[0014] The present invention also provides a method for preparing the aspartase variant, comprising culturing a host cell containing the encoding nucleic acid, and then recovering the aspartase variant from the cell culture. The host cell containing the encoding nucleic acid is capable of constitutively expressing or inducibly expressing the aspartase variant.

[0015] The present invention further provides the use of the aforementioned aspartic acidase variant or the aforementioned encoding nucleic acid or the aforementioned recombinant vector, expression cassette or recombinant cell in the preparation of a product having catalytic ammonia-addition activity to acrylic acid.

[0016] The present invention further provides the use of the aspartate enzyme variant, the encoded nucleic acid, or the recombinant vector, expression cassette, or recombinant cell in the production of the target product β-alanine or its derivatives.

[0017] The target product is selected from β-alanine, β-alanine salt and / or β-alanine polymers.

[0018] The present invention provides a method for producing a target product, comprising the following steps: reacting acrylic acid and an ammonia-containing material under the catalysis of the aforementioned aspartate enzyme variant to obtain the target product β-alanine or a derivative thereof.

[0019] Specifically, the target product is any one or more of the following: β-alanine, β-alanine salt, and β-alanine polymer.

[0020] The aspartate enzyme variant obtained through extensive research and screening in this invention has significantly increased enzyme activity under the same conditions compared to wild-type aspartate enzyme, by at least 28 times and up to 81 times. Therefore, it can improve the conversion rate of acrylic acid in the ammonia reaction of acrylic acid and has greater practical value in the production of β-alanine or its derivatives. Detailed Implementation

[0021] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0022] Example 1: Obtaining the mutant Based on previous mutant studies, this invention, through further research and exploration, discovered that wild-type aspartic acidase derived from Bacillus exists in the amino acid sequence shown in SEQ ID NO: 1. Based on mutations including T187I / M321I / K324I / N326C, the addition of the I103F mutation enhances its activity. Further additions of D20V, I41L, H42V, P43V, N142V, L156F, H188A, L258I, A422T, or Y429D further improve its activity, indicating that it can enhance the catalytic activity and conversion efficiency of acrylic acid ammonia addition.

[0023] The resulting mutants are shown in Table 1. Based on the wild-type gene sequence (SEQ ID NO: 2), the coding genes of the corresponding mutants were synthesized according to the designed mutation sites.

[0024] Example 2: Construction of recombinant strains containing mutants Using the vector plasmid pET21a as a vector and the mutant-encoding gene from Example 1 as a template, the corresponding nucleotide gene fragment was amplified by PCR using primers designed based on the mutation site. Then, the PCR-amplified gene fragment and pET21a were mixed in a 1:1 molar ratio, and a plasmid containing the aspartate enzyme or its variant encoding gene was constructed using Gibson assembly. Using *Escherichia coli* BL21(DE3) as the host cell, the recombinant plasmid containing the aspartate enzyme encoding gene was introduced into the host cell to obtain a recombinant strain containing the corresponding mutant.

[0025] Example 3: Expression of mutants 1. The recombinant strain obtained in Example 2 was cultured using self-induction medium. The composition of the self-induction medium was as follows: peptone 10 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 3.55 g / L, potassium dihydrogen phosphate 3.4 g / L, ammonium chloride 2.68 g / L, sodium sulfate 0.71 g / L, magnesium sulfate heptahydrate 0.493 g / L, ferric chloride hexahydrate 0.27 g / L, 100% glycerol 20 mL / L, glucose 0.5 g / L, lactose 2 g / L, and ampicillin sodium 50 mg / L.

[0026] The recombinant strain was inoculated into self-induction medium and fermented in a batch fermentation manner. The mixture was cultured with shaking at 30°C and 200 rpm for 20 hours.

[0027] 2. Cell collection: The following three methods can be used: ① Centrifugation: Centrifuge the recombinant strain cell culture medium at 4000g for 10 minutes and collect the cells; ② Hollow fiber membrane filtration: Collect the cells using a 0.22-micron hollow fiber membrane; ③ Ceramic membrane filtration: Collect the cells using a 50kDa ceramic membrane.

[0028] Cells are preferably collected by centrifugation under laboratory conditions (this method is used in this embodiment).

[0029] 3. Purification of expressed proteins: Resuspend cells in ultrapure water until the OD550 value is 200. Disrupt cells using sonication under the following conditions: 1 second on, 2 second off, 180W, for a total of 20-25 minutes. Alternatively, cells can be lysed using high-pressure homogenization under the following conditions: 50Hz, 800 bar, for two cycles.

[0030] Cell debris and macromolecular impurities were removed by high-speed centrifugation under the following conditions: 10-50 mL of cell lysis buffer was centrifuged at 12000 rpm for 4°C for 20-40 min. The supernatant after removing cell debris and macromolecular impurities was then concentrated using ultrafiltration centrifugation. Amicon Ultra-15 (or 50) mL ultrafiltration tubes equipped with Ultracel-50 ultrafiltration membranes were used, and the centrifugation conditions were: 12000 rpm for 4°C for 20-40 min. The aspartate protein retained in the ultrafiltration tube was resuspended in 1-5 mL of 4°C pure water.

[0031] 4. Determination of enzyme protein concentration: The aspartate enzyme protein resuspension was diluted 100-500 times and its concentration was determined using the Bradford method.

[0032] Example 4: Determination of the catalytic activity of acrylic acid ammonia addition 1. Enzyme activity detection methods 25 mg of crude enzyme protein of wild-type aspartic acid enzyme or its variant was mixed with 1.0 mL of 400 mM pH 8.0 substrate, and the mixture was stirred at 40 °C for 1.0 h. The activity of the enzyme in catalyzing acrylic acid ammonia addition was then determined.

[0033] The substrate preparation method is as follows: The substrate for the aspartic acid enzyme variant is prepared by using 400 mM acrylic acid or acrylate and adjusting the pH to 8.0 with ammonia.

[0034] 2. Liquid Chromatography Detection Method High-performance liquid chromatography (HPLC) was used to detect the consumption of substrate in the reaction system. The detection conditions and methods are as follows: Column: Agilent 5 AQ-C18 column; Mobile phase: Weigh 1.44 g of potassium dihydrogen phosphate, dissolve it in ultrapure water, and bring the volume to 1000 mL; adjust the pH to 2.6 with phosphoric acid (approximately 400 μL), filter using a 0.22 μm mixed filter membrane, measure 100 mL of methanol solution, mix the two solutions at a volume ratio of 9:1, and sonicate the resulting solution for 30 min to remove air bubbles; Preparation of potassium dihydrogen phosphate at pH 2.6: Dissolve 1.44 g of KH₂PO₄ in 1 L of ultrapure water, and adjust the pH to 2.6 with 0.6-0.7 mL of 85% phosphoric acid. Injection volume: 10 µl; Detection wavelength: 210nm; Elution conditions: constant elution rate of 1.0 mL / min.

[0035] 3. Definition of terms The activity of catalytic ammonia-additase is defined as the amount of enzyme required to catalyze the conversion of 1 mM substrate per minute under specified conditions, which is defined as 1 U. The activity of catalytic ammonia-additase per unit cell is defined as the amount of enzyme required to catalyze the conversion of 1 mM substrate per minute at a cell concentration of OD550 under specified conditions, defined as 1 U / OD550. Relative enzyme activity: the ratio of the enzyme activity of aspartic acid enzyme variant to that of wild-type aspartic acid enzyme.

[0036] The relative enzyme activities of the aspartate enzyme variants are shown in Table 1, which are the average values ​​obtained from three experiments.

[0037] Table 1. Mutation sites of aspartate enzyme variants and relative activities of catalytic ammonia-additase. ; ;

[0038] Variants containing multiple alterations are separated by slashes (" / "), for example: "A422T / Y429D" represents mutations at positions 422 and 429, where amino acid A at position 422 is replaced by T and amino acid Y at position 429 is replaced by D.

[0039] The invention described and claimed herein is not limited to the specific embodiments disclosed herein, as the above embodiments are intended to illustrate a particular aspect of the invention and are intended to include any equivalent embodiments within the scope of the invention.

Claims

1. An aspartate enzyme variant, characterized in that, It has aspartate enzyme activity, and its amino acid sequence has more than 98% homology with SEQ ID NO:

1. It also has the I103F / T187I / M321I / K324I / N326C mutation based on SEQ ID NO:

1. Or, based on this, at least one of the following mutations may also exist: D20V, I41L, H42V, P43V, N142V, L156F, H188A, L258I, A422T and / or Y429D.

2. The aspartate enzyme variant as described in claim 1, characterized in that, The aspartic acidase variant exhibits improved catalytic activity for ammonia addition to acrylic acid.

3. The aspartate enzyme variant as described in claim 2, characterized in that, In addition to the I103F / T187I / M321I / K324I / N326C mutations present in SEQ ID NO: 1, any of the following mutations also exist: D20V; I41L; H42V; P43V; D20V / L156F; D20V / A422T; D20V / Y429D; D20V / I41L / L156F; D20V / I41L / H188A; D20V / I41L / A422T; D20V / I41L / Y429D; D20V / H42V / P43V; D20V / H42V / N142V; D20V / N142V / L156F; D20V / L156F / H188A; D20V / L156F / A422T; D20V / H188A / L258I; D20V / L258I / A422T; D20V / A422T / Y429D; D20V / I41L / H42V / L156F; D20V / I41L / N142V / H188A; D20V / I41L / L258I / A422T; D20V / I42L / L156F / H188A; D20V / P43V / L156F / Y429D; D20V / P43V / A422T / Y429D; D20V / N142V / L156F / L258I; D20V / N142V / H188A / L258I; D20V / N142V / A422T / Y429D; D20V / L156F / H188A / A422T; D20V / L156F / A422T / Y429D; D20V / I41L / H42V / P43V / H188A; D20V / I41L / H42V / L156F / L258I; D20V / H42V / N142V / L156F / A422T; D20V / P43V / N142V / H188A / L258I; D20V / P43V / H188A / L258I / A422T; N142V; L156F; H188A; L258I; H42V / N142V; P43V / L156F; N142V / L258I; I41L / H42V / L156F; I41L / H42V / H188A; I41L / P43V / N142V; I41L / P43V / Y429D; I41L / N142V / A422T; I41L / N142V / H188A; I41L / L156F / L258I; I41L / L156F / Y429D; I41L / L258I / A422T; H42V / P43V / Y429D; H42V / N142V / L156F; H42V / L156F / H188A; H42V / L258I / A422T; I41L / H42V / P43V / Y429D; I41L / H42V / P43V / L258I; I41L / H42V / N142V / Y429D; I41L / H42V / H188A / A422T; I41L / N142V / L156F / L258I; I41L / H188A / L258I / Y429D; H42V / P43V / L156F / A422T; H42V / N142V / L156F / H188A; H42V / N142V / L258I / A422T; H42V / L156F / L258I / Y429D; H42V / H188A / L258I / A422T; D20V / N142V / L258I / A422T / Y429D; D20V / N142V / L156F / H188A / Y429D; I41L / H42V / P43V / N142V / L156F; I41L / H42V / P43V / H188A / A422T; I41L / P43V / N142V / L156F / L258I; A422T; Y429D; N142V / L156F; L258I / A422T; A422T / Y429D; P43V / N142V / L258I; P43V / L156F / Y429D; P43V / H188A / A422T; P43V / L258I / A422T; P43V / A422T / Y429D; N142V / L156F / L258I; L156F / H188A / L258I; L156F / H188A / Y429D; L156F / L258I / A422T; H188A / L258I / Y429D; H188A / A422T / Y429D; P43V / N142V / L156F / A422T; P43V / N142V / H188A / L258I; P43V / H188A / L258I / Y429D; N142V / L156F / H188A / A422T; N142V / L156F / A422T / Y429D; N142V / L156F / L258I / Y429D; N142V / H188A / A422T / Y429D; L156F / L258I / A422T / Y429D; L156F / H188A / L258I / A422T; I41L / N142V / H188A / A422T / Y429D; I41L / L156F / H188A / L258I / A422T; H42V / N142V / L156F / L258I / Y429D; P43V / N142V / H188A / L258I / A422T; N142V / L156F / L258I / A422T / Y429D; D20V / I41L / H42V / P43V / N142V / L258I; D20V / P43V / L156F / H188A / L258I / Y429D; I41L / H42V / N142V / L156F / L258I / A422T; I41L / P43V / L156F / L258I / A422T / Y429D; P43V / N142V / L156F / L258I / A422T / Y429D; I41L / H42V / L156F / H188A / L258I / A422T / Y429D.

4. The nucleic acid encoding the aspartate enzyme variant as described in any one of claims 1 to 3.

5. A recombinant vector, expression cassette, transgenic cell line, or recombinant cell containing the nucleic acid encoding as described in claim 4.

6. A method for preparing the aspartase variant according to any one of claims 1 to 3, comprising the steps of culturing a host cell containing the nucleic acid encoding the as described in claim 4, and then recovering the aspartase variant from the cell culture.

7. The use of the aspartate enzyme variant according to any one of claims 1 to 3, or the nucleic acid encoding variant according to claim 4, or the recombinant vector, expression cassette, or recombinant cell according to claim 5 in the preparation of a product having catalytic ammonia-addition activity to acrylic acid.

8. The use of the aspartate enzyme variant according to any one of claims 1 to 3, the nucleic acid encoding variant according to claim 4, or the recombinant vector, expression cassette, or recombinant cell according to claim 5 in the production of the target product β-alanine or a derivative thereof.

9. The application according to claim 8, wherein the target product is selected from: β-alanine, β-alanine salt and / or polymers of β-alanine.

10. A method for producing a target product, comprising the steps of: reacting acrylic acid and an ammonia-containing material under the catalysis of an aspartate enzyme variant according to any one of claims 1 to 3 to obtain the target product β-alanine or a derivative thereof; Specifically, the target product is any one or more of the following: β-alanine, β-alanine salt, and β-alanine polymer.

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