L-threonine aldolase mutants and their use in catalyzing the synthesis of l-serine derivatives

Abstract

The present application provides an L-threonine aldolase mutant, the amino acid sequence of which is obtained by mutating a wild-type L-threonine aldolase as shown in SEQ ID NO. 1, the mutation comprising at least one mutation in the amino acids at positions 185, 198, 281, 8, 142, 171 of SEQ ID NO. 1. The present application also provides the use of the mutant as a catalyst in the synthesis of (2 S ,3 R )‑2‑amino‑3‑hydroxy‑3‑(4‑methylsulfonylphenyl)propionic acid. Compared with other biological catalysts, the L-threonine aldolase mutant of the present application has high catalytic activity, high yield and high de value up to 99%, and has the advantages of high atom economy, high optical purity of product, mild reaction condition, environmental friendliness, simple product post-treatment, etc., and is one of the most ideal reactions for the synthesis of (2 S ,3 R )‑2‑amino‑3‑hydroxy‑3‑(4‑methylsulfonylphenyl)propionic acid.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and enzyme catalysis technology, specifically relating to L-threonine aldolase (L-TA) mutants and their encoding genes, recombinant expression vectors, recombinant expression transformants, and their applications in the catalytic synthesis of L-serine derivatives, particularly for the efficient and highly selective synthesis of (2 S ,3 R )-2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid. Background Technology

[0002] L-serine and its derivatives are important chiral compounds widely used in drug synthesis, food additives, and fine chemical production. Among them, (2) S ,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid, as a promising chiral intermediate, has shown significant application value in the pharmaceutical field. Traditional chemical synthesis methods often suffer from problems such as cumbersome steps, low atom economy, harsh reaction conditions, difficulty in chiral control, and environmental pollution. For example, chemical synthesis typically requires the use of toxic reagents, heavy metal catalysts, or complex chiral induction strategies, and the optical purity of the products is often insufficient to meet the demands of high-end applications.

[0003] In recent years, biocatalysis has become an ideal alternative route for synthesizing chiral compounds due to its advantages such as mild reaction conditions, environmental friendliness, high regioselectivity, and stereoselectivity. L-threonine aldolase (L-TA) can catalyze the aldol condensation reaction between glycine and aldehydes to generate chiral L-serine derivatives, making it a highly promising biocatalyst. However, wild-type L-TA typically suffers from limitations such as low catalytic activity, insufficient product yield, and unsatisfactory stereoselectivity (de value) when catalyzing non-natural substrates (such as p-4-methanesulfonylbenzaldehyde), restricting its industrial application.

[0004] To address these issues, protein engineering, particularly site-directed mutagenesis, has been widely applied to modify enzyme performance, aiming to improve catalytic efficiency, stability, and stereoselectivity for non-natural substrates. While mutation studies targeting L-TA have been reported, mutants that simultaneously achieve high catalytic activity, high yield, and a de value as high as 99% are still relatively scarce, especially in the synthesis of (2... S ,3 R In this specific application, 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid is used.

[0005] Therefore, it is necessary to develop an L-TA mutant with excellent catalytic performance to achieve (2) S , 3RThe efficient and high-optical-purity green synthesis of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid has significant industrial application value and scientific research significance. Based on in-depth analysis of the L-TA structure, this invention rationally designs or directionally evolves its key amino acid sites, successfully obtaining a series of mutants with significantly improved catalytic performance, providing an effective solution to address the efficiency and selectivity issues existing in current technologies. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an L-threonine aldolase mutant that can catalyze the synthesis of (2) glycine and p-4-methanesulfonylbenzaldehyde. S ,3 R )-2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: The first object of the present invention is to provide an L-threonine aldolase mutant whose amino acid sequence is obtained by mutating the wild-type L-threonine aldolase as shown in SEQ ID NO.1, said mutation comprising at least one of the amino acids 185, 198, 281, 8, 142, and 171 of SEQ ID NO.1.

[0008] Preferably, the mutation includes at least one of the following A1-A6 mutations of SEQ ID NO.1: A1): The 185th position mutates from E to C; A2): The 198th position mutates from I to V; A3): The 281st position is mutated from S to T; A4): The 8th position mutates from Y to C; A5): The 142nd position is mutated from E to any one of A, G, V, R, and K; A6): The 171st position is mutated from A to any one of G, R, and K; Preferably, the mutation is any one of the following (1)-(17): (1) The amino acid residue at position 185 of SEQ ID NO.1 is mutated from E to C; (2) The amino acid residue at position 185 of SEQ ID NO.1 is mutated from E to C; the amino acid residue at position 198 is mutated from I to V; and the amino acid residue at position 281 is mutated from S to T. (3) The 8th amino acid residue of SEQ ID NO.1 is mutated from Y to C; the 142nd amino acid residue is mutated from E to G; the 171st amino acid residue is mutated from A to R; the 185th amino acid residue is mutated from E to C; and the 198th amino acid residue is mutated from I to V. (4) The 8th amino acid residue of SEQ ID NO.1 is mutated from Y to C; the 142nd amino acid residue is mutated from E to G; the 171st amino acid residue is mutated from A to R; the 185th amino acid residue is mutated from E to C; the 198th amino acid residue is mutated from I to V; and the 281st amino acid residue is mutated from S to T. (5) The amino acid residue at position 198 of SEQ ID NO.1 is mutated from I to V; (6) The amino acid residue at position 281 of SEQ ID NO.1 is mutated from S to T; (7) The 8th amino acid residue in SEQ ID NO.1 is mutated from Y to C; (8) The amino acid residue at position 142 of SEQ ID NO.1 is mutated from E to A; (9) The amino acid residue at position 142 of SEQ ID NO.1 is mutated from E to G; (10) The amino acid residue at position 142 of SEQ ID NO.1 is mutated from E to V; (11) The amino acid residue at position 142 of SEQ ID NO.1 is mutated from E to R; (12) The amino acid residue at position 142 of SEQ ID NO.1 is mutated from E to K; (13) The amino acid residue at position 171 of SEQ ID NO.1 is mutated from A to G; (14) The amino acid residue at position 171 of SEQ ID NO.1 is mutated from A to R; (15) The amino acid residue at position 171 of SEQ ID NO.1 is mutated from A to K; (16) The 8th amino acid residue in SEQ ID NO.1 is mutated from Y to C; the 142nd amino acid residue is mutated from E to G; (17) The 8th amino acid residue of SEQ ID NO.1 is mutated from Y to C; the 142nd amino acid residue is mutated from E to G; and the 171st amino acid residue is mutated from A to R.

[0009] A second objective of this invention is to provide an enzyme preparation comprising the aforementioned L-threonine aldolase mutant.

[0010] A third objective of this invention is to provide a nucleic acid encoding the aforementioned L-threonine aldolase mutant.

[0011] Preferably, the nucleic acid includes at least one of the following B1-B6 mutations of SEQ ID NO.2: B1): Nucleotides 553-555 are mutated from GAA to TGT; B2): Nucleotides 592-594 are mutated from ATT to GTG; B3): Nucleotides 841-843 are mutated from AGC to ACC; B4): Nucleotides 22-24 are mutated from TAC to TGT; B5): Nucleotides 424-426 are mutated from GAA to any one of GCA, GGT, GTT, CGT, or AAG; B6): Nucleotides 511-513 are mutated from GCG to any one of GGT, CGT, or AAG; Preferably, the mutation is any one of the following (1)-(17): (1) Nucleotides at positions 553-555 are mutated from GAA to TGT; (2) Nucleotides at positions 553-555 are mutated from GAA to TGT; nucleotides at positions 592-594 are mutated from ATT to GTG; nucleotides at positions 841-843 are mutated from AGC to ACC; (3) Nucleotides at positions 22-24 are mutated from TAC to TGT; nucleotides at positions 424-426 are mutated from GAA to GGT; nucleotides at positions 511-513 are mutated from GCG to CGT; nucleotides at positions 553-555 are mutated from GAA to TGT; nucleotides at positions 592-594 are mutated from ATT to GTG; (4) Nucleotides at positions 22-24 are mutated from TAC to TGT; nucleotides at positions 424-426 are mutated from GAA to GGT; nucleotides at positions 511-513 are mutated from GCG to CGT; nucleotides at positions 553-555 are mutated from GAA to TGT; nucleotides at positions 592-594 are mutated from ATT to GTG; nucleotides at positions 841-843 are mutated from AGC to ACC; (5) Nucleotides at positions 592-594 are mutated from ATT to GTG; (6) Nucleotides at positions 841-843 are mutated from AGC to ACC; (7) Nucleotides at positions 22-24 are mutated from TAC to TGT; (8) Nucleotides 424-426 are mutated from GAA to GCA; (9) Nucleotides 424-426 are mutated from GAA to GGT; (10) Nucleotides 424-426 are mutated from GAA to GTT; (11) Nucleotides 424-426 are mutated from GAA to CGT; (12) Nucleotides 424-426 are mutated from GAA to any of AAG; (13) Nucleotides at positions 511-513 are mutated from GCG to GGT; (14) Nucleotides at positions 511-513 are mutated from GCG to CGT; (15) Nucleotides 511-513 are mutated from GCG to AAG; (16) Nucleotides at positions 22-24 are mutated from TAC to TGT; nucleotides at positions 424-426 are mutated from GAA to GGT; (17) The nucleotides at positions 22-24 are mutated from TAC to TGT; the nucleotides at positions 424-426 are mutated from GAA to GGT; and the nucleotides at positions 511-513 are mutated from GCG to CGT.

[0012] A fourth objective of this invention is to provide an expression vector comprising the aforementioned nucleic acid; Preferably, the expression vector is pET28a.

[0013] A fifth object of the present invention is to provide a cell comprising the above-described nucleic acid or expression vector; Preferably, the cells are bacteria such as Escherichia coli or fungi.

[0014] A sixth objective of this invention is to provide a method for preparing the above-mentioned L-threonine aldolase mutant, the method comprising introducing the above-mentioned expression vector into cells, culturing the cells, collecting bacterial cells, and obtaining the L-threonine aldolase mutant; or The preparation method includes directly culturing the above-mentioned cells, collecting bacterial cells, and obtaining the L-threonine aldolase mutant. Preferably, the cells are bacteria such as Escherichia coli or fungi; As a further preferred embodiment, the preparation method further includes the following steps: breaking the bacterial cells, centrifuging to collect the supernatant, and obtaining crude L-threonine aldolase solution.

[0015] The seventh objective of this invention is to provide the application of the above-mentioned L-threonine aldolase mutant, enzyme preparation, nucleic acid, expression vector, and cell as catalysts in the synthesis of L-serine derivatives; using glycine and 4-methanesulfonylbenzaldehyde as substrates; Preferably, the L-serine derivative is (2 S ,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid; Preferably, 4-methanesulfonylbenzaldehyde, pyridoxal phosphate, glycine, the above-mentioned L-threonine aldolase mutant or enzyme preparation or nucleic acid or expression vector or cells are mixed evenly with an inorganic solvent and reacted to obtain (2) S ,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid; the inorganic solvent is a KH2PO4-K2HPO4 buffer solution; Preferably, the inorganic solvent has a pH value of 6.5-8.5, and more preferably, the inorganic solvent has a pH value of 8.0; Preferably, the reaction temperature is 25-45°C, and more preferably, the reaction temperature is 35°C. Preferably, the concentration ratio of glycine to 4-methanesulfonylbenzaldehyde is 1:1 to 10:1, and more preferably, the ratio is 6:1. Preferably, the amount of pyridoxal phosphate added is 0.1%-1% of the molar concentration of 4-methanesulfonylbenzaldehyde, more preferably 0.75%; Preferably, the concentration of the 4-methanesulfonylbenzaldehyde is 10-400 mM; more preferably, it is 400 mM. Preferably, the amount of crude L-threonine aldolase added is 0.125-10 mg / mL, and more preferably, the amount of crude L-threonine aldolase added is 8 mg / mL. Preferably, the reaction time is 1-48 h; more preferably, the reaction time is 48 h.

[0016] The eighth object of the present invention is to provide a (2) S ,3 R The synthesis method of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid involves mixing 4-methanesulfonylbenzaldehyde, pyridoxal phosphate, glycine, the above-mentioned L-threonine aldolase mutant or enzyme preparation or nucleic acid or expression vector or cells with an inorganic solvent, and reacting to obtain (2) S ,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid; the inorganic solvent is a KH2PO4-K2HPO4 buffer solution; Preferably, the inorganic solvent has a pH value of 6.5-8.5, and more preferably, the inorganic solvent has a pH value of 8.0; Preferably, the reaction temperature is 25-45°C, and more preferably, the reaction temperature is 35°C. Preferably, the concentration ratio of glycine to 4-methanesulfonylbenzaldehyde is 1:1 to 10:1, and more preferably, the ratio is 6:1. Preferably, the amount of pyridoxal phosphate added is 0.1%-1% of the molar concentration of 4-methanesulfonylbenzaldehyde, more preferably 0.75%; Preferably, the concentration of the 4-methanesulfonylbenzaldehyde is 10-400 mM; more preferably, it is 400 mM. Preferably, the amount of crude L-threonine aldolase added is 0.125-10 mg / mL, and more preferably, the amount of crude L-threonine aldolase added is 8 mg / mL. Preferably, the reaction time is 1-48 h; more preferably, the reaction time is 48 h.

[0017] The L-threonine aldolase mutant of the present invention can act as a catalyst to catalyze the synthesis of (2) glycine and 4-methanesulfonylbenzaldehyde as substrates. S ,3 R )-2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid. Compared with other biocatalysts, the L-threonine aldolase mutant of this invention exhibits high catalytic activity, high yield, and meets the requirement of a de value as high as 99%. It also possesses advantages such as high atom economy, high product optical purity, mild reaction conditions, environmental friendliness, and simple product post-processing. It is (2 S ,3 R It is one of the most ideal reactions for the synthesis of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Plate (a), seed culture (b), fermentation broth (c), and crude enzyme solution (d) for mutant a29.

[0019] Figure 2 SDS-Page protein electrophoresis of mutant a29.

[0020] Figure 3 For product (2) S ,3 R Synthetic route of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid.

[0021] Figure 4 In the middle, figure a shows the racemic product (3) R The liquid chromatogram of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid, Figure b shows the product (2) prepared under optimal reaction conditions. S ,3 RHigh performance liquid chromatogram of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid. Detailed Implementation

[0022] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0023] The raw materials used in the preparation of this invention are as follows: pET28a vector: purchased from Novagen, catalog number: 69864-3.

[0024] Escherichia coli BL21(DE3): Purchased from Beijing TransGen Biotechnology Co., Ltd., catalog number: CD601-02.

[0025] LB medium: Each liter contains 10g tryptone, 5g yeast extract, and 5g NaCl. Adjust the pH to 7.4 with 1mL of 1mol / L NaOH, and bring the volume to 1L with deionized water. Autoclave for 20 min.

[0026] TB medium: Each liter contains 12 g tryptone, 24 g yeast extract, 4 mL 87% glycerol, and 100 mL phosphate buffer (pH 6.5), and is brought to a final volume of 1 L with deionized water. Autoclave for 20 min.

[0027] Example 1: Screening and Determination of Mutation Sites For those derived from the genus *Pelosinus* ( Pelosinus sp. IPA-1The wild-type L-threonine aldolase (WP_285718658.1) protein (its amino acid sequence is shown in SEQ ID NO.1, and the codon-optimized (E. coli preferred) nucleic acid sequence (gene) is shown in SEQ ID NO.2 (synthesized by Qingke Biotechnology, codon optimization software: JCat: http: / / www.jcat.de) was subjected to sequence analysis, mutation, and functional verification. 22 amino acid sites were specifically selected (8th, 11th, 86th, 142nd, 171st, 185th, 197th, 198th, 202nd, 205th, 279th, 281st, 85th, 140th, 177th, 192nd, 200th, 288th, 323rd, 163rd, 189th, 201st) for further analysis (using molecular docking: Autodock software for molecular docking simulation; Pymol). Software for visualizing the three-dimensional structures of proteins and small molecules (obtained through Hotsport hotspot amino acid analysis) screened 12 important amino acid sites (amino acids 8, 11, 86, 142, 171, 185, 197, 198, 202, 205, 279, and 281) from 22 amino acid sites. These 12 amino acid sites were then mutated in different ways to obtain mutant proteins.

[0028] Derived from the genus Pelosinus ( Pelosinus sp. IPA-1 The amino acid sequence of the wild-type L-threonine aldolase (WP_285718658.1) is as follows (SEQ ID No. 1): MYSFKNDYSEGAHPKILHALIETNLEQVEGYGEDYYTRKAVELLKENIKKKDIDIHLFSGGTQTNLTALSAFLRPHEAAIAANTGHILVHETGAIEAIGHKIISIEVQDGKIGPEHLKTVLETHADEHMVKPKLVYISNPTEIGSIYKKKELEKLSQFCRENQLFLYVDGAR LGSALCSNENDMELSDLAMLVDAFYIGGTKNGALMGEALVICRDSLKEDFRFHMKQKGALLAKGRLLGIQFLELFRDGLYFNLATHANEMASLLRGEISQAGYLFLTHSSPNQIFPILPNEIITKLQEKYSFYIWSQVDSEHSAIRLVTSWATKEDDVWKFIEDLKGLCNKK.

[0029] The nucleotide sequence obtained by codon optimization of the above amino acid sequence is as follows (SEQ ID No. 2):

[0030] Table 1. Mutation sites and nucleotide sequences corresponding to the mutants.

[0031] Example 2 Construction of recombinant plasmids and recombinant bacteria I. Construction of Wild-Type Recombinant Expression Vectors Using homologous recombination, and employing the primers listed in Table 2, the recombinase gene sequence of SEQ ID No. 2 was used as a template. The PCR procedure in Table 3 was followed to amplify the target fragment containing the mutation site. After column purification using a column purification kit, the obtained target fragment was inserted into the pET28a vector. EcoR I and Xho Between the I restriction sites, the recombinant expression vector pET28a-AmDH was obtained, and sequencing confirmed its correctness.

[0032] Table 2 Primers for a1-a24 single point mutations

[0033]

[0034] Table 3 PCR amplification program

[0035] II. Construction of mutant recombinant expression vectors The nucleotide sequences encoding mutants a1–a29 (see Table 1) were inserted into the pET28a vector. EcoR I and Xho Between the I restriction sites, recombinant expression vectors pET28a-1 to pET28a-29 were obtained, and sequencing confirmed their correctness.

[0036] III. Preparation of Recombinant Bacteria 1. The recombinant expression vector pET28a-AmDH obtained in step one and the recombinant expression vectors pET28a-1 to pET28a-29 obtained in step two were transformed into Escherichia coli BL21(DE3) host bacteria, respectively, to obtain wild-type recombinant bacteria and mutant recombinant bacteria (numbered sequentially as mutant recombinant bacteria a1 to a29). The specific steps are as follows: (1) Take a 2 mL sterilized EP tube, add 50 μL of E. coli BL21(DE3) competent cells (thawed on an ice bath beforehand) and 1-5 μL of recombinant plasmid containing the target gene. After gently mixing, place the EP tube in an ice bath for 30 min. Then, heat shock it in a 42 ℃ water bath for 45-60 s. After the heat shock, quickly transfer the EP tube to an ice bath and let it stand for 2 min.

[0037] (2) Continue to add 500 μL of sterile LB medium (without antibiotics) to competent cells, mix well, and then incubate at 37 ℃ and 200 rpm for 1 h to allow the cells to recover.

[0038] (3) Take 50-200 μL of transformed competent cells and add them to LB agar medium containing kanamycin (final concentration 50 μg / mL). Spread the cells evenly with a spreader and place the plate at 37 ℃ until the liquid is completely absorbed. Then, invert the plate and incubate overnight at 37 ℃.

[0039] (4) Select a single colony from the plating plate. Figure 1 a) Inoculate the culture into 10 mL of LB medium containing kanamycin (final concentration 50 μg / mL). Incubate the centrifuge tubes overnight at 37 ℃ and 180 rpm for 12–16 h. Subsequently, prepare glycerol-containing bacteria and store them at -80 ℃.

[0040] 2. Induction and expression of recombinant bacterial strains: The specific steps are as follows. (1) Preparation of seed culture: 10 μL of glycerol bacteria was inoculated into 10 mL of LB medium (containing kanamycin, final concentration 50 μg / mL), and cultured overnight at 37 ℃ and 180 rpm for 12-16 h. Figure 1 b).

[0041] (2) Scale-up culture: The seed culture was transferred to 50 mL of TB scale-up culture medium at an inoculation rate of 1%, and cultured at 37 ℃ and 180 rpm. When OD 600 When the value reaches 0.6-0.8, add 50 μL of the inducer IPTG (final concentration 0.5 μM), and then induce expression at 20 ℃ for 20 h. Figure 1 c). After cultivation, the fermentation broth was centrifuged at 6000 rpm for 10 min, and the cells were collected. The cell pellet was then washed three times with PBS buffer (50 mM, pH 7.5) to remove the culture medium. The cells were resuspended in PBS and homogenized using a high-pressure homogenizer at 4 °C. The cells were then centrifuged at 12000 rpm for 30 min, and the supernatant was collected to obtain the crude enzyme solution. Figure 1 d).

[0042] 3. Purification and concentration determination of target proteins a1~a29 Ni-NTA affinity chromatography was used to separate and purify target proteins containing the 6×His tag. First, the Ni-NTA column was equilibrated with protein loading buffer, and then the filtered crude enzyme solution was loaded onto the Ni-NTA column. Next, a gradient elution was performed using protein purification elution buffers with imidazole concentrations of 30 mM, 60 mM, 300 mM, and 500 mM. After desalting, the purified protein was obtained. Protein concentration was determined using the Bradford method. The purified concentrations of different target proteins ranged from 10.3 to 12.4 mg / mL, with the concentration of the target protein from different batches of a29 culture being 11.9 ± 0.4 mg / mL.

[0043] 4. Determination of the molecular weight of the target protein: The molecular weight of the target protein was analyzed using SDS-PAGE electrophoresis. The specific procedure was as follows: The protein sample was mixed with a loading buffer containing SDS and β-mercaptoethanol and heated to denature the protein and uniformly charge it with a negative charge. Then, the lower separating gel and the upper stacking gel were poured sequentially into the glass plate interlayer, and a sample comb was inserted to form sample wells. After the gel solidified, it was placed in the electrophoresis tank, buffer was added, and the prepared sample and protein standard (marker) were added to the wells. After power was turned on, the sample was first concentrated into a narrow line in the stacking gel under low voltage, and then the voltage was switched to higher voltage to separate the proteins in the separating gel according to their molecular weight until the indicator reached the bottom of the gel. After electrophoresis, the gel was removed and stained with Coomassie Brilliant Blue to reveal the protein bands. The background was then removed with destaining solution, and finally, the gel was photographed using a gel imaging system. The molecular weight of the target protein was analyzed based on the migration position of the protein markers. The molecular weight of the target protein a29 was approximately 45 kDa. Figure 2 ).

[0044] Figure 1 Plate (a), seed culture (b), fermentation broth (c), and crude enzyme solution (d) for mutant a29.

[0045] Figure 2 SDS-Page protein electrophoresis of mutant a29.

[0046] Example 3: Enzyme activity assay of L-threonine aldolase mutant protein The reaction system consisted of 2 mL KH₂PO₄-K₂HPO₄ (50 mM, pH 7.5): 100 mM glycine, 10 mM 4-methanesulfonylbenzaldehyde, 5 μM pyridoxal phosphate (PLP), and 0.125 mg·mL⁻¹ -1 The target protein (wild-type recombinant bacterial protein or mutant recombinant bacterial protein prepared in Example 2) was reacted at 35 °C with shaking at 200 rpm for 1 h. After the reaction, the product (2) was obtained. S,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid. After the reaction was complete, the reaction system was boiled for 10 min, then centrifuged at 10,000 × g for 5 min. The supernatant was diluted with 50 mM KH₂PO₄-K₂HPO₄ (pH 8.0) buffer. Then, it was filtered through a 0.22 μm aqueous filter and placed in a liquid chromatography vial.

[0047] Product yield and stereoselectivity detection conditions: Product concentration and diastereoselectivity (de) were detected using a C18 column and an Agilent-1260 high-performance liquid chromatography (HPLC) pre-column derivatization system. The HPLC detection conditions were as follows: mobile phase volume ratio: KH₂PO₄-K₂HPO₄ (50 mM, pH 8.0):acetonitrile = 80:20; flow rate: 0.5 mL / min. -1 Temperature: 40 °C; Running time: 40 min.

[0048] Figure 3 For product (2) S ,3 R Synthetic route of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid.

[0049] Relative enzyme activity calculation: The system with the highest conversion rate is taken as 100%. Stereoselectivity is not detected for systems with relative enzyme activity below 10%.

[0050] Table 4 Statistical results of enzyme activity

[0051] Table 4 shows that there are significant differences in enzyme activity among the different mutants, with mutant a29 exhibiting the highest enzyme activity and stereoselectivity. The mutant a29 was sequenced by Qingke Biotechnology, and the sequencing was successful. Subsequent experiments will be conducted using mutant a29.

[0052] Example 4 (2) S ,3 R Synthesis of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid Initial reaction conditions: The reaction system consisted of 2 mL KH₂PO₄-K₂HPO₄ (50 mM, pH 7.5): 100 mM glycine, 10 mM 4-methanesulfonylbenzaldehyde, 0.1 mM PLP, and 0.125 mg·mL⁻¹ -1The target protein (mutant a29 protein) was reacted at 25 °C with shaking at 200 rpm for 48 h. After the reaction, the reaction system was boiled for 10 min, then centrifuged at 10,000 × g for 5 min. The supernatant was diluted with 50 mM KH2PO4-K2HPO4 (pH 8.0) buffer. Then, it was filtered through a 0.22 μm aqueous filter and placed in a liquid chromatography vial.

[0053] The reactions were carried out under different reaction conditions, as detailed in Table 5.

[0054] Product yield and stereoselectivity detection conditions: Product concentration and diastereoselectivity (de) were detected using a C18 column and an Agilent-1260 high-performance liquid chromatography (HPLC) pre-column derivatization system. The HPLC detection conditions were as follows: mobile phase volume ratio: KH₂PO₄-K₂HPO₄ (50 mM, pH 8.0):acetonitrile = 80:20; flow rate: 0.5 mL / min. -1 Temperature: 40 °C; Running time: 40 min.

[0055] Tests showed that the product (2) could be obtained under different reaction conditions. S ,3 R )-2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid, all with de values ​​of 99%, and specific yields are shown in Table 5.

[0056] Table 5. Under different reaction conditions (2) S ,3 R Synthesis of 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid

[0057] As shown in Table 5, the optimal result is at sequence number 28. At this point, although the product yield is less than 90%, the product yield is the highest due to the significantly increased substrate concentration.

[0058] That is, the reaction system is 2 mL KH2PO4-K2HPO4 (50 mM, pH 8.0): 2400 mM glycine, 400 mM 4-methanesulfonylbenzaldehyde, 3 mM PLP, and 10 mg·mL -1The target protein (mutant a29 protein) was detected. The reaction was carried out at 35 °C with shaking at 200 rpm for 48 h. After the reaction, the reaction system was boiled for 10 min, then centrifuged at 10,000 × g for 5 min. The supernatant was diluted with 50 mM KH₂PO₄-K₂HPO₄ (pH 8.0) buffer. The solution was then filtered through a 0.22 μm aqueous filter and placed in a HPLC vial for detection by high-performance liquid chromatography (HPLC). The results are shown below. Figure 4 .

[0059] Figure 4 In the middle, figure a shows the racemic product (3) R Figure b shows the high performance liquid chromatogram of the product prepared under the optimal reaction conditions (corresponding to serial number 28).

[0060] Depend on Figure 4 It can be seen that the product obtained after the reaction is (2) S ,3 R )-2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid, with a de value of 99%.

[0061] Example 5: Thermal stability of the L-threonine aldolase of the present invention Heat incubation conditions: After incubating the crude enzyme solution of mutant a29 in a water bath at 50°C for a period of time, the following reaction was performed and its remaining activity was detected.

[0062] Reaction conditions: The reaction system consisted of 2 mL KH₂PO₄-K₂HPO₄ (50 mM, pH 8.0): 100 mM glycine, 10 mM 4-methanesulfonylbenzaldehyde, 0.1 mM PLP, and 0.125 mg·mL⁻¹ -1 The target protein (mutant a29 protein) was reacted at 35 °C with shaking at 200 rpm for 1 h. After the reaction, the reaction system was boiled for 10 min, then centrifuged at 10,000 × g for 5 min. The supernatant was diluted with 50 mM KH2PO4-K2HPO4 (pH 8.0) buffer. Then, it was filtered through a 0.22 μm aqueous filter and placed in a liquid chromatography vial.

[0063] Yield and stereoselectivity detection conditions: Product concentration and diastereoselectivity (de) were detected using a C18 column and an Agilent-1260 high-performance liquid chromatography (HPLC) pre-column derivatization system. The HPLC detection conditions were as follows: mobile phase: KH₂PO₄-K₂HPO₄ (50 mM, pH 8.0):acetonitrile = 80:20; flow rate: 0.5 mL / min. -1Temperature: 40 °C; Running time: 40 min. The test results are shown in Table 6.

[0064] Relative activity calculation: The conversion rate before incubation is 100%.

[0065] Table 6. Thermal stability of the L-threonine aldolase of the present invention

[0066] Example 6: pH stability of the L-threonine aldolase of the present invention Incubation conditions: The crude enzyme solution of mutant a29 was incubated in a buffer solution at pH 5.0 for a period of time, and its remaining activity was detected after the reaction. The reaction process and detection method were the same as in Example 5. The detection results are shown in Table 7.

[0067] Table 7 pH stability of the L-threonine aldolase of the present invention

[0068] Example 7 Storage stability of the L-threonine aldolase of the present invention Storage conditions: After incubating the crude enzyme solution in a refrigerator at 4°C for a period of time, the remaining activity was detected after the reaction. The reaction process and detection method were the same as in Example 5. The detection results are shown in Table 8.

[0069] Table 8 Storage stability of the L-threonine aldolase of the present invention

[0070] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An L-threonine aldolase mutant, characterized by: Its amino acid sequence was obtained by mutating the wild-type L-threonine aldolase shown in SEQ ID NO.1, wherein the mutation is any one of the following (1)-(4): (1) The amino acid residue at position 185 of SEQ ID NO.1 is mutated from E to C; (2) The amino acid residue at position 185 of SEQ ID NO.1 is mutated from E to C; the amino acid residue at position 198 is mutated from I to V; and the amino acid residue at position 281 is mutated from S to T. (3) The 8th amino acid residue of SEQ ID NO.1 is mutated from Y to C; the 142nd amino acid residue is mutated from E to G; the 171st amino acid residue is mutated from A to R; the 185th amino acid residue is mutated from E to C; and the 198th amino acid residue is mutated from I to V. (4) The 8th amino acid residue of SEQ ID NO.1 is mutated from Y to C; the 142nd amino acid residue is mutated from E to G; the 171st amino acid residue is mutated from A to R; the 185th amino acid residue is mutated from E to C; the 198th amino acid residue is mutated from I to V; and the 281st amino acid residue is mutated from S to T.

2. An enzyme preparation, characterized in that: The enzyme preparation includes the L-threonine aldolase mutant as described in claim 1.

3. The nucleic acid encoding the L-threonine aldolase mutant of claim 1.

4. The nucleic acid according to claim 3, characterized in that: The nucleic acid is any one of the mutations in (1)-(4) of SEQ ID NO.2 below: (1) Nucleotides at positions 553-555 are mutated from GAA to TGT; (2) Nucleotides at positions 553-555 are mutated from GAA to TGT; nucleotides at positions 592-594 are mutated from ATT to GTG; nucleotides at positions 841-843 are mutated from AGC to ACC; (3) Nucleotides at positions 22-24 are mutated from TAC to TGT; nucleotides at positions 424-426 are mutated from GAA to GGT; nucleotides at positions 511-513 are mutated from GCG to CGT; nucleotides at positions 553-555 are mutated from GAA to TGT; nucleotides at positions 592-594 are mutated from ATT to GTG; (4) Nucleotides at positions 22-24 are mutated from TAC to TGT; nucleotides at positions 424-426 are mutated from GAA to GGT; nucleotides at positions 511-513 are mutated from GCG to CGT; nucleotides at positions 553-555 are mutated from GAA to TGT; nucleotides at positions 592-594 are mutated from ATT to GTG; nucleotides at positions 841-843 are mutated from AGC to ACC.

5. An expression vector comprising the nucleic acid of claim 3 or 4.

6. The expression vector according to claim 5, characterized in that: The expression vector is pET28a.

7. A cell comprising the nucleic acid of claim 3 or 4 or the expression vector of claim 5 or 6.

8. The cell according to claim 7, characterized in that: The cells are bacteria.

9. The cell according to claim 7, characterized in that: The cells are either Escherichia coli or fungi.

10. A method for preparing the L-threonine aldolase mutant according to claim 1, characterized in that: The preparation method includes introducing the expression vector according to claim 5 or 6 into cells, culturing the cells, collecting bacterial cells, and obtaining the L-threonine aldolase mutant; or The preparation method includes directly culturing the cells according to any one of claims 7 to 9, collecting the bacterial cells, and obtaining the L-threonine aldolase mutant.

11. The preparation method according to claim 10, characterized in that: The cells are bacteria.

12. The preparation method according to claim 10, characterized in that: The cells are either Escherichia coli or fungi.

13. The preparation method according to claim 10, characterized in that: The preparation method further includes the following steps: breaking the bacterial cells, centrifuging to collect the supernatant, and obtaining crude L-threonine aldolase solution.

14. The use of the L-threonine aldolase mutant of claim 1, the enzyme preparation of claim 2, the nucleic acid of claim 3 or 4, the expression vector of claim 5 or 6, and the cell of any one of claims 7 to 9 as a catalyst in the synthesis of L-serine derivatives; characterized in that: Glycine and 4-methanesulfonylbenzaldehyde were used as substrates.

15. The application according to claim 14, characterized in that: The L-serine derivative is (2) S ,3 R )-2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid.

16. The application according to claim 15, characterized in that: 4-Methanesulfonylbenzaldehyde, pyridoxal phosphate, glycine, the L-threonine aldolase mutant of claim 1, or the enzyme preparation of claim 2, or the nucleic acid of claim 3 or 4, or the expression vector of claim 5 or 6, or the cells of any one of claims 7 to 9, are mixed evenly with an inorganic solvent and reacted to obtain (2) S ,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid; the inorganic solvent is a KH2PO4-K2HPO4 buffer solution.

17. The application according to claim 16, characterized in that: The inorganic solvent has a pH value of 6.5-8.

5.

18. The application according to claim 17, characterized in that: The inorganic solvent has a pH value of 8.

0.

19. The application according to claim 16, characterized in that: The reaction temperature is 25-45℃.

20. The application according to claim 19, characterized in that: The reaction temperature is 35°C.

21. The application according to claim 14, characterized in that: The concentration ratio of glycine to 4-methanesulfonylbenzaldehyde is 1:1 to 10:

1.

22. The application according to claim 21, characterized in that: The concentration ratio of glycine to 4-methanesulfonylbenzaldehyde is 6:

1.

23. The application according to claim 16, characterized in that: The amount of pyridoxal phosphate added is 0.1%-1% of the molar concentration of 4-methanesulfonylbenzaldehyde.

24. The application according to claim 23, characterized in that: The amount of pyridoxal phosphate added is 0.75% of the molar concentration of 4-methanesulfonylbenzaldehyde.

25. The application according to claim 16, characterized in that: The concentration of the 4-methanesulfonylbenzaldehyde is 10-400 mM.

26. The application according to claim 25, characterized in that: The concentration of the 4-methanesulfonylbenzaldehyde is 400 mM.

27. The application according to claim 16, characterized in that: The amount of crude L-threonine aldolase solution added is 0.125-10 mg / mL.

28. The application according to claim 27, characterized in that: The crude L-threonine aldolase solution was added at a concentration of 8 mg / mL.

29. The application according to claim 16, characterized in that: The reaction time is 1-48 h.

30. The application according to claim 29, characterized in that: The reaction time is 48 h.

31. A kind of (2) S ,3 R A method for synthesizing 2-amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid, characterized in that: 4-Methanesulfonylbenzaldehyde, pyridoxal phosphate, glycine, the L-threonine aldolase mutant of claim 1, or the enzyme preparation of claim 2, or the nucleic acid of claim 3 or 4, or the expression vector of claim 5 or 6, or the cells of any one of claims 7 to 9, are mixed evenly with an inorganic solvent and reacted to obtain (2) S ,3 R 2-Amino-3-hydroxy-3-(4-methanesulfonylphenyl)propionic acid; the inorganic solvent is a KH2PO4-K2HPO4 buffer solution.

32. The synthesis method according to claim 31, characterized in that: The inorganic solvent has a pH value of 6.5-8.

5.

33. The synthesis method according to claim 32, characterized in that: The inorganic solvent has a pH value of 8.

0.

34. The synthesis method according to claim 31, characterized in that: The reaction temperature is 25-45℃.

35. The synthesis method according to claim 34, characterized in that: The reaction temperature is 35°C.

36. The synthesis method according to claim 31, characterized in that: The concentration ratio of glycine to 4-methanesulfonylbenzaldehyde is 1:1 to 10:

1.

37. The synthesis method according to claim 36, characterized in that: The concentration ratio of glycine to 4-methanesulfonylbenzaldehyde is 6:

1.

38. The synthesis method according to claim 31, characterized in that: The amount of pyridoxal phosphate added is 0.1%-1% of the molar concentration of 4-methanesulfonylbenzaldehyde.

39. The synthesis method according to claim 38, characterized in that: The amount of pyridoxal phosphate added is 0.75% of the molar concentration of 4-methanesulfonylbenzaldehyde.

40. The synthesis method according to claim 31, characterized in that: The concentration of the 4-methanesulfonylbenzaldehyde is 10-400 mM.

41. The synthesis method according to claim 40, characterized in that: The concentration of the 4-methanesulfonylbenzaldehyde is 400 mM.

42. The synthesis method according to claim 31, characterized in that: The amount of crude L-threonine aldolase solution added is 0.125-10 mg / mL.

43. The synthesis method according to claim 42, characterized in that: The crude L-threonine aldolase solution was added at a concentration of 8 mg / mL.

44. The synthesis method according to claim 31, characterized in that: The reaction time is 1-48 h.

45. The synthesis method according to claim 44, characterized in that: The reaction time is 48 h.

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