L-threonine aldolase mutant R318L / H128N / N90Q and application thereof

Abstract

The invention relates to L-threonine aldolase, in particular to an L-threonine aldolase mutant R318L / H128N / N90Q and application of the L-threonine aldolase mutant R318L / H128N / N90Q. The amino acid sequence of the L-threonine aldolase mutant is shown as SEQ ID NO: 3, and the L-threonine aldolase mutant is obtained by changing the 318th amino acid of L-threonine aldolase of which the amino acid sequence is SEQ ID NO: 1 into Leu from Arg, changing the 128th amino acid into Asn from His and changing the 90th amino acid into Gln from Asn. The mutant has high catalytic activity in a reaction of catalyzing benzaldehyde and derivatives thereof and glycine to synthesize beta-hydroxyl-alpha-amino acid, when a whole cell expressing R318L / H128N / N90Q is used for catalysis, the diastereoselectivity (de) of benzaldehyde and glycine to synthesize benzene serine is 65.8%, and the yield is 67.7%; the diastereoselectivity (de) of p-fluorobenzene serine synthesized from p-fluorobenzaldehyde and glycine is 77.5%, and the yield of p-fluorobenzene serine is 80.3%. The mutant has application potential in biosynthesis of beta-hydroxyl-alpha-amino acid.

Description

Technical Field

[0001] The invention relates to threonine aldolase, and in particular to an L-threonine aldolase mutant R318L / H128N / N90Q and an application thereof. Background Art

[0002] β-Hydroxy-α-amino acid is an important chiral intermediate, widely used in the fields of fine chemicals and medicine. Phenylserine, the representative structure of β-hydroxy-α-amino acid, is a widely used intermediate in chemical synthesis and enzymatic reactions. At the same time, phenylserine is also an important active fragment of many natural products, such as: ecumicin, a cyclic peptide with strong inhibitory activity against Mycobacterium tuberculosis, lysobactin, a natural cyclic peptide antibiotic with antibacterial activity, cyclomarin A, which has strong inhibitory activity against Mycobacterium tuberculosis and Plasmodium falciparum, nyuzenamideC with anti-angiogenic activity, and gymnangiamide, a pentapeptide with anticancer activity. In addition, L-threo-3,4-dihydroxyphenylserine (droxidopa) is a drug for the treatment of Parkinson's disease, L-threo-4-nitrophenylserine is an intermediate in the synthesis of the antibiotic chloramphenicol, L-threo-4-methylsulfonylphenylserine is an intermediate in the synthesis of the antibiotics florfenicol and thiamphenicol, and L-threo-4-hydroxyphenylserine is an important intermediate in the formation of the antibiotics vancomycin and teicoplanin.

[0003] The industrial production of chiral β-hydroxy-α-amino acids relies on chiral resolution, which has cumbersome steps and low atomic utilization. Its asymmetric synthesis usually requires expensive metal catalysts, which are costly and difficult to apply in actual production. Therefore, the enzymatic synthesis of β-hydroxy-α-amino acids has attracted much attention. L-threonine aldolase can catalyze the synthesis of β-hydroxy-α-amino acids containing two chiral centers from simple raw materials glycine and substrate aldehyde in one step. The reaction conditions are mild, simple and efficient. However, the C α The stereoselectivity can reach more than 99%, but for C β The stereoselectivity of L-threonine aldolase is only moderate or poor, which limits its application in asymmetric synthesis. Therefore, the construction of L-threonine aldolase with high stereoselectivity and high catalytic yield is the key to the enzymatic synthesis of β-hydroxy-α-amino acids. Summary of the invention

[0004] In order to develop a threonine aldolase with high selectivity, the present invention compares the similarities and differences between the wild-type L-threonine aldolase (amino acid sequence as shown in SEQ ID NO: 1) isolated from black bear feces samples and homologous enzyme proteins from multiple angles and multiple systems from primary structure to higher-level structure. In the early stage, it was determined that the sites affecting the biological properties of the wild-type L-threonine aldolase were the 318th and 128th amino acids, and the arginine (Arg) at position 318 was mutated to leucine (Leu), and the histidine (His) at position 128 was mutated to asparagine (Asn). Under the premise, the 90th amino acid was discovered through semi-rational design and rational design, and it was mutated from asparagine (Asn) to glutamine (Gln), and an enzyme mutant was obtained, named L-TA R318L / H128N / N90Q mutant. The coding gene (SEQ ID NO: 4) of the L-TA R318L / H128N / N90Q mutant was obtained by molecular cloning technology. The L-TA R318L / H128N / N90Q mutant enzyme protein was obtained by constructing a GST fusion expression vector of the mutant gene and introducing it into the genetic engineering bacteria E. coli BL21 for induction of expression. The L-TA R318L / H128N / N90Q mutant was used as a catalyst, benzaldehyde and its various substituted derivatives as substrates, glycine as a cosubstrate, and 5-pyridoxal phosphate as a coenzyme, and the enzyme catalyzed reaction was carried out under appropriate conditions and media. The results showed that the mutant also had generally high catalytic activity for benzaldehyde derivatives. When benzaldehyde was used as a substrate, the mutant synthesized phenylserine with a diastereoselectivity of 65.8% and a yield of 67.7%; when p-fluorobenzaldehyde was used as a substrate, the mutant synthesized p-fluorophenylserine with a diastereoselectivity of 77.5% and a yield of 80.3%. In summary, this mutant has application value in the industrial production of β-hydroxy-α-amino acids.

[0005] The present invention provides an L-threonine aldolase mutant, characterized in that its amino acid sequence is as shown in SEQ ID NO: 3, which is obtained by changing the 318th amino acid of the L-threonine aldolase of the amino acid sequence of SEQ ID NO: 1 from Arg to Leu, the 128th amino acid from His to Asn, and the 90th amino acid from Asn to Gln.

[0006] The gene encoding the L-threonine aldolase mutant also belongs to the protection scope of the present invention.

[0007] In some preferred embodiments of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0008] The expression cassette, vector or recombinant bacteria containing the gene also fall within the protection scope of the present invention.

[0009] The vector provided by the present invention can be a cloning vector, comprising the L-TA R318L / H128N / N90Q mutant gene and other elements required for plasmid replication; or it can be an expression vector, comprising the L-TA R318L / H128N / N90Q mutant gene and other elements that enable the successful expression of the protein. In some embodiments of the present invention, the expression vector is a pGEX-6p-2 vector into which the L-TAR318L / H128N / N90Q mutant gene is inserted, and the pGEX-6p-2 vector is a known commercially available vector.

[0010] The recombinant bacteria provided by the present invention may be a recombinant bacteria comprising a cloning vector, such as E. coli DH5α, and the L-TA R318L / H128N / N90Q mutant gene may be replicated by culturing the recombinant bacteria; or it may be a recombinant bacteria comprising an expression vector, such as E. coli BL21, and the recombinant bacteria may be cultured under appropriate conditions and protein expression may be performed, for example: an appropriate amount of IPTG is added to the culture fluid of the recombinant bacteria, and the expression of the L-TA R318L / H128N / N90Q mutant protein is induced at 16°C.

[0011] The invention also provides a method for preparing the L-threonine aldolase mutant, comprising the following steps: synthesizing the coding gene of the L-threonine aldolase mutant, constructing an expression vector, transforming a protein expression host bacterium, inducing protein expression and purifying the protein.

[0012] In a preferred embodiment of the preparation method of the present invention, the nucleotide sequence of the gene encoding the L-threonine aldolase mutant is shown in SEQ ID NO:4.

[0013] In some embodiments of the preparation method of the present invention, the expression vector is a pGEX-6p-2 vector, and the protein expression host bacteria is E. coli BL21 (DE3).

[0014] The present invention also provides a catalyst, the active ingredient of which comprises the L-threonine aldolase mutant. The catalyst can be used alone or simultaneously with other suitable catalysts to improve the diastereoselectivity of the enzyme or to carry out multiple catalytic reactions in the same reaction system.

[0015] The use of the L-threonine aldolase mutant or the catalyst in the aldol condensation reaction also belongs to the protection scope of the present invention.

[0016] In some preferred embodiments of the present invention, the L-threonine aldolase mutant or the catalyst is used to carry out a catalytic reaction with benzaldehyde and a series of substituted derivatives thereof as substrates, glycine as a cosubstrate, and pyridoxal-5-phosphate as a coenzyme to synthesize phenylserine and its derivatives.

[0017] In some preferred embodiments of the present invention, the catalytic reaction is carried out at 15-37° C. and pH 5.0-11.0.

[0018] The L-TA R318L / H128N / N90Q mutant of the present invention can catalyze the reversible aldol condensation reaction of benzaldehyde and its substituted derivatives and glycine under the reaction conditions of 15-37°C and pH 5.0-11.0. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 .Schematic diagram of the synthesis of β-hydroxy-α-amino acids catalyzed by L-threonine aldolase (L-TA).

[0020] Figure 2 .SDS-PAGE electrophoresis of L-threonine aldolase mutant R318L / H128N / N90Q; wherein M is the protein molecular weight standard (Marker); R318L / H128N / N90Q is a mutant protein with a molecular weight of approximately 38.7KD. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific examples. It should be noted that the following examples are only for explanation and illustration and do not limit the scope of the present invention in any way.

[0022] Main reagents and consumables:

[0023] Prime STAR Max Premix (2×) (Cat. No. R045A), BamH I restriction endonuclease (Cat. No. 1010S), Xho I restriction endonuclease (Cat. No. 1094S), T4 DNA Ligase (Cat. No. D2011A), and 10× T4 Ligase buffer were all purchased from Bao Biotechnology Co., Ltd. (Dalian);

[0024] The pGEX-6p-2 plasmid is a known E. coli expression vector, alias pGEX6P2, pGEX6P2, vector size 4985 bp, Tac promoter, vector tag N-GST, vector resistance Ampicillin (ampicillin), purchased from Shanghai Shenggong Biotechnology Co., Ltd., and also preserved in this laboratory;

[0025] Trans5α competent cells (Cat. No. CD201-01) and E. coli BL21 (DE3) competent cells (Cat. No. CD601) were purchased from Quanshijin Biotechnology Co., Ltd.;

[0026] Glutathione Sepharose 4B, purchased from GE Healthcare, catalog number 10223836;

[0027] PreScission Protease, purchased from Gen Script, catalog number Z02799-100;

[0028] Bradford protein concentration assay kit, purchased from Beyotime, catalog number P0006;

[0029] Phenylserine standard (CAS: 101246-66-6, molecular formula: C 20 H 23 N3O2), 1-heptanesulfonic acid sodium salt (CAS: 22767-50-6, molecular formula: C7H 15 NaO3S), 1,4-dioxane (CAS: 123-91-1, molecular formula: C4H8O2) were purchased from J&K Technology Co., Ltd.;

[0030] Glycine (CAS: 56-40-6, molecular formula: C2H5NO2, item number A502065), benzaldehyde (CAS: 100-52-7, molecular formula: C7H6O, item number B802740), 5-pyridoxal phosphate (English name PLP, CAS: 41468-25-1, molecular formula C8H 12 NO7P, Catalog No. A610455) were purchased from Shanghai Biotechnology Co., Ltd.

[0031] LB medium

[0032] Each 100 mL of LB medium contains: 1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride, pH 7.4.

[0033] Preparation method: Dissolve 10g tryptone, 5g yeast extract, and 10g sodium chloride in 950mL ddH2O, then adjust the pH to 7.4 with NaOH and dilute to 1L with dd H2O. If preparing solid culture medium, add 15g agar per liter. Sterilize with high pressure steam at 121℃ for 20min.

[0034] PBS buffer

[0035] Preparation of pH 7.4, 10mM PBS: weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve in 800mL distilled water, adjust the pH value of the solution to 7.4 with HCl, and finally add distilled water to 1L. Sterilize at 121℃ high pressure steam for at least 20 minutes, and store at room temperature or in a 4℃ refrigerator for later use.

[0036] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and reference can be made to the relevant experimental manuals or manufacturer's instructions.

[0037] Example 1. Preparation of L-threonine aldolase R318L / H128N / N90Q mutant

[0038] 1. Design of L-threonine aldolase mutants

[0039] The wild-type L-threonine aldolase gene (L-TA gene, SEQ ID NO: 2) was isolated from the fecal sample of a healthy black bear in the Sichuan Black Bear Protection and Incubation Base by this laboratory. The open reading frame of the L-TA gene is 1032 bp in length, and the L-threonine aldolase encoded by it consists of 343 amino acids. The isolation and cloning process of the gene is recorded in the patent application with application number 2019109657806, publication number CN110592058A, and invention name "Threonine aldolase, its encoding gene and its application in droxidopa biosynthesis", and the entire contents of the patent application are included in this article by reference.

[0040] The amino acid sequence (343aa) of the wild-type L-threonine aldolase is:

[0041] MYSFKNDYSEGAHPRILETLLRTNLEQCEGYGKDTYCEEAENLIKNKLNNESIEVHFISGGTQTNLIAISAFLRPHEGVISADTGHIFVNEAGSIEATGHKVISVDVVDGKLRRDDILSVLSKFTNEHVVKPKLVYISNSTEIGTIYKKSELEELSKVCRENNLLLFMDGARLGSALSCKENDLTLEDISKLTDAFYIGGTKNGALLGEALVICNKDLQEDFRYHLKQKGAMLAKGRLLGIQFIELFKDDLFFEIGKHENDMADILRDGISRLGYEFLVDSPSNQIFPVFNNDIIRELEKNYGFNIWEKVNEEKTAIRLVTSFATKEEPCLEFIKFLSGLTNK (SEQ ID NO: 1) The nucleotide sequence (1032 bp) of the gene encoding the wild-type L-threonine aldolase is:

[0042]

[0043] We compared the similarities and differences between wild-type L-threonine aldolase and homologous enzyme proteins from multiple angles and multiple layers from primary structure to higher-order structure, and determined that the sites that affect the enzymatic properties are amino acids 318, 128, and 90 of the wild-type L-threonine aldolase, and the corresponding nucleotide sequences are codons 952-954, 382-384, and 268-270. The codons at positions 952-954 of the wild-type L-TA gene sequence were changed from AGA to CTG, the codons at positions 382-384 were changed from CAT to AAT, and the codons at positions 268-270 were changed from AAT to CAA, thereby replacing the original arginine (R) at position 318 of the amino acid sequence with leucine (L), the original histidine (H) with asparagine (N), and the original asparagine (N) with glutamine (Q), to obtain an enzyme mutant named L-TA R318L / H128N / N90Q mutant, whose amino acid sequence is shown in SEQ ID NO:3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:4.

[0044] 2. Obtaining the L-TA R318L / H128N / N90Q mutant gene

[0045] The L-TA R318L / H128N / N90Q mutant gene can be obtained by whole gene synthesis or molecular cloning. In this experiment, the mutant gene was obtained by PCR.

[0046] 1. Primer design

[0047] Primers were designed for the obtained L-TA R318L / H128N / N90Q mutant gene sequence (SEQ ID NO: 4), and the restriction site BamH I (GGATCC) was added to the 5' end primer, and the restriction site Xho I (CTCGAG) was added to the 3' end primer. The nucleotide sequence of the primer is as follows:

[0048] Upstream primer L-TA-R318L-BamH IF:

[0049] 5′-CGCGGATCCATGTATAGTTTTAAAAATGATTAT-3′ (SEQ ID NO: 5),

[0050] Downstream primer L-TA-R318L-Xho IR:

[0051] 5'-CCGCTCGAGTTATTTATTAGTTAATCCACTTA-3' (SEQ ID NO: 6).

[0052] Upstream primer L-TA-R318L / H128N-BamH IF:

[0053] 5′-TAAGTTTACTAATGAGAATGTTGTAAAACCAAAGCTTG-3′ (SEQ ID NO:7)

[0054] Downstream primer L-TA-R318L / H128N-Xho IR:

[0055] 5′-CAAGCTTTGGTTTTACAACATTCTCATTAGTAAACTTAC-3′(SEQ ID NO:8)

[0056] Upstream primer L-TA-R318L / H128N / N90Q-BamH IF:

[0057] 5′-GGCATATATTTGTACAAGAAGCAGGTTCAATAGA-3′(SEQ ID NO:9)

[0058] Downstream primer L-TA-R318L / H128N / N90Q-Xho IR:

[0059] 5′-GTTGCTTCTATTGAACCTGCTTCTTTGTACAAATATA-3′(SEQ ID NO:10)

[0060] The above primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0061] 2. L-TA R318L / H128N / N90Q mutant gene cloning

[0062] Using the plasmid of the L-TA mutant R318L / H128N gene obtained in the previous laboratory work as a template, the upstream primers and downstream primers obtained in step 1 were used to amplify the L-TA R318L / H128N / N90Q mutant gene according to the following PCR system and procedure.

[0063] PCR system: Prime STAR Max Premix (2×) 25 μL, plasmid template 0.5 μL, upstream primer L-TAR318L / H128N / N90Q-BamH IF (10 μM) 2 μL, downstream primer L-TA-R318L / H128N / N90Q-Xho IR (10 μM) 2 μL, add ddH2O to a total volume of 50 μL.

[0064] The PCR program is as follows:

[0065] a. Pre-denaturation at 94℃ for 5 min;

[0066] b. Denaturation at 98°C for 10 sec, annealing at 47°C for 10 sec, and extension at 72°C for 10 sec; 40 cycles;

[0067] c. Extend at 72°C for 10 min.

[0068] The PCR amplification product was detected by 1.2% agarose gel electrophoresis to obtain a target band of about 1000 bp in size. The target band was cut under ultraviolet light, and the L-TA R318L / H128N / N90Q mutant gene fragment was recovered using Omega Gel Extraction Kit D2500 according to the kit instructions.

[0069] 3. Expression vector construction

[0070] (1) Enzyme digestion and ligation

[0071] BamH I and Xho I restriction endonucleases were used to double-digest the L-TA R318L / H128N / N90Q mutant gene and pGEX-6p-2 vector, respectively. Enzyme digestion system (gene): 35 μL of L-TA R318L / H128N / N90Q mutant gene, 3 μL of BamH I enzyme, 3 μL of Xho I enzyme, 6 μL of 10×K buffer, and sterile double distilled water was added to the system to 60 μL. Enzyme digestion system (vector): 2 μL of pGEX-6p-2 vector, 0.5 μL of BamH I enzyme, 0.5 μL of Xho I enzyme, 1 μL of 10×K buffer, and sterile double distilled water was added to the system to 10 μL. Enzyme digestion conditions: 37°C for 3 hours.

[0072] Then, T4 DNA ligase was used to connect the L-TA R318L / H128N / N90Q mutant gene and pGEX-6p-2 linear vector after restriction digestion: 6 μL of L-TA R318L / H128N / N90Q mutant gene after restriction digestion, 2 μL of pGEX-6p-2 linear vector, 1 μL of T4 DNA Ligase, and 1 μL of 10×T4 Ligase buffer. The ligation was carried out at 16°C overnight to obtain the ligation product pGEX-6p-2 / L-TA R318L / H128N / N90Q.

[0073] (2) Conversion

[0074] Place Trans5α competent cells (full gold, CD201-01) on ice, add 10μL pGEX-6p-2 / L-TA R318L / H128N / N90Q after the cells thaw, and place on ice for 30 minutes. Place in a 42℃ water bath for 90 seconds, then place on ice for 2 minutes. Add 600μL sterile LB liquid culture medium, and culture at 37℃, 150rpm shaking for 45 minutes. Pipette 200μL of the cultured bacterial solution, spread on Amp+ resistance (100μg / mL) LB plate culture medium, and invert and culture at 37℃ overnight.

[0075] (3) Positive clone screening

[0076] Pick a single colony on the LB plate, inoculate it in Amp+ resistance (100 μg / mL) LB liquid medium, culture at 37°C, 220 rpm shaking to OD 600≈1.0, and collect the bacteria by centrifugation at 8000 rpm for 5 min for plasmid extraction. Use OMEGAPlasmid Mini Kit I (Cat. No.: D6943) to extract the plasmid according to the instructions, and perform double enzyme digestion identification on the plasmid.

[0077] Enzyme digestion system:

[0078]

[0079] Enzyme digestion conditions: 37°C for 3h. 1.2% agarose gel electrophoresis was used to detect the digestion products, and the recombinant plasmids with correct double digestion identification were sent to TAKARA (Dalian, China) for sequencing. The recombinant plasmids with correct sequencing results were used as the expression vectors of L-TAR318L / H128N / N90Q mutants.

[0080] 4. GST fusion heterologous expression of enzyme protein

[0081] (1) Plasmid transformation into E. coli BL21 (DE3) cells

[0082] Remove the competent cells of E.coli BL21 (DE3) from -80℃ and place them on ice. After the cells melt, add 10μL of the expression vector pGEX-6p-2 / L-TA R318L / H128N / N90Q with the correct sequencing results and place them on ice for 30 minutes. Heat shock at 42℃ for 90s, then place them on ice for 2 minutes. Add 600μL of sterile LB liquid culture medium and culture at 37℃, 150rpm shaking for 45 minutes. Pipette 200μL of the cultured bacterial solution, spread it on the Amp+ resistance (100μg / mL) LB plate culture medium, and invert and culture it at 37℃ overnight. Pick a single colony on the LB plate, perform colony PCR identification according to the PCR system and procedure in step 2, and use the colony with the correct identification result as the protein expression strain.

[0083] (2) Protein expression and purification

[0084] a. Inoculate the protein expression strain into sterile LB liquid culture medium with a final concentration of ampicillin of 50 μg / mL and culture at 37°C and 170 rpm.

[0085] b. Wait for OD 600 When the pH value was ≈0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was induced overnight (12 h) at 16°C and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 5 min.

[0086] c. Resuspend the bacteria in the ratio of 1L culture system plus 30mL lysis buffer (pH 7.4, 10mM PBS), and use an ultra-high pressure nano homogenizer to break the cell wall at 4℃. Divide the broken bacterial solution into 50mL sterile centrifuge tubes precooled at 4℃, centrifuge at 4℃, 12000rpm for 20min, and transfer the supernatant to a 50mL sterile centrifuge tube precooled at 4℃ with a precision pipette after the centrifugation is completed.

[0087] d. Glutathione Sepharose 4B filler (GE Healthcare) was loaded into a chromatography column (GE Healthcare) at a ratio of 5 mL filler per liter of culture system. Three column volumes were washed with 4°C precooled pH 7.4, 10 mM sterile PBS to remove anhydrous ethanol. The supernatant obtained in step c was combined with Glutathione Sepharose 4B and suspended vertically at 4°C for 3 h.

[0088] e. After binding, centrifuge at 500 rpm for 5 min to precipitate the filler. Rinse the filler with 4°C precooled pH 7.4, 10 mM sterile PBS for 3-5 column volumes to remove impurities.

[0089] f. Add 4°C pre-cooled enzyme digestion buffer (pH 7.4, 10 mM PBS), add PreScission Protease (GenScript, Z02799-100), and digest at 4°C overnight.

[0090] g. After the enzyme digestion is completed, the supernatant is released from the chromatography column to obtain L-TAR318L / H128N / N90Q enzyme solution.

[0091] h. The L-TA R318L / H128N enzyme solution was subjected to SDS-PAGE detection to identify its molecular weight. The concentration of the L-TA R318L / H128N / N90Q enzyme solution was determined using a Bradford protein concentration determination kit (Beyotime, P0006) according to the kit instructions.

[0092] The results are as follows Figure 2 As shown, the SDS-PAGE test results showed that the L-TA R318L / H128N / N90Q mutant was successfully expressed in a soluble form, the protein molecular weight was about 38.7KD, the enzyme protein purity was very high, and it was a single band. It was determined that the concentration of the L-TA R318L / H128N / N90Q enzyme solution was 2mg / mL.

[0093] Example 2. Synthesis of phenylserine using L-TA R318L / H128N / N90Q mutant and detection of de value

[0094] 1. Synthesis of phenylserine

[0095] In a reaction buffer containing 25% DMF (v / v) in Gly-NaOH (pH=8.0, glycine concentration of 100 mM), 40 mM substrate benzaldehyde, 400 mM glycine, 400 mg / mL whole cells expressing the L-TA mutant, and 50 μM PLP were added. The reaction was carried out at 37°C for 3 h. The product was subjected to centrifugal ultrafiltration using a 10 kDa ultrafiltration tube. The lower liquid sample obtained after ultrafiltration was lyophilized, and then the corresponding organic phase solvent was added to make up the volume to an appropriate concentration.

[0096] 2. Detection of phenylserine and calculation of de value

[0097] The sample obtained after ultrafiltration in step 1 was diluted 10 times to achieve better HPLC separation effect. The amount of L-threo-phenylserine and its diastereoisomer L-erythro-phenylserine in the sample was detected by HPLC. HPLC instrument model: Agilent 1260; parameter setting: chromatographic column: CHIRALPAK*ZWIX(+) chiral column (250×3mm); wavelength 225nm, column temperature 35°C; mobile phase: methanol (25mM formic acid + 25mM diethylamine): pure water = 98:2 as mobile phase, flow rate 0.5mL / min. According to the same HPLC parameters, the L-threo-phenylserine standard (0.1mg / mL, pH 7.4, 10mM PBS) was detected to make a standard curve for the quantification of the reaction product. The de value (diastereoisomericexcess, diastereomeric excess percentage) was determined based on the following equation:

[0098] [(the amount of L-threo-phenylserine - the amount of L-erythro-phenylserine) / (the amount of L-threo-phenylserine + the amount of L-erythro-phenylserine)]×100%.

[0099] Example 3. Synthesis of p-fluorophenylserine using L-TA R318L / H128N / N90Q mutant and detection of de value

[0100] 1. Synthesis of 4-fluorophenylserine

[0101] In a reaction buffer containing 25% DMF (v / v) in Gly-NaOH (pH=8.0, glycine concentration of 100 mM), 40 mM substrate p-fluorobenzaldehyde, 400 mM glycine, 400 mg / mL whole cells expressing the L-TA mutant, and 50 μM PLP were added. The reaction was carried out at 37°C for 3 h. The product was subjected to centrifugal ultrafiltration using a 10 kDa ultrafiltration tube. The lower liquid sample obtained after ultrafiltration was lyophilized, and then the corresponding organic phase solvent was added to make up the volume to an appropriate concentration.

[0102] 2. Detection of fluorophenylserine and calculation of de value

[0103] The sample obtained after ultrafiltration in step 1 was diluted 10 times to achieve better HPLC separation effect. The amount of L-threo-p-fluorophenylserine and its diastereoisomer L-erythro-p-fluorophenylserine in the sample was detected by HPLC. HPLC instrument model: Agilent 1260. Parameter setting: Chromatographic column: CHIRALPAK*ZWIX(+) chiral column (250×3mm); wavelength 225nm, column temperature 35°C; mobile phase: methanol (25mM formic acid + 25mM diethylamine): pure water = 98:2 as mobile phase, flow rate 0.5mL / min. The L-threo-p-fluorophenylserine standard (0.1mg / mL, pH 7.4, 10mM PBS) was detected according to the same HPLC parameters, and a standard curve was made for the quantification of the reaction product. The de value (diastereoisomericexcess, diastereomeric excess percentage) was determined based on the following equation:

[0104] [(the amount of L-threo-p-fluorophenylserine-the amount of L-erythro-p-fluorophenylserine) / (the amount of L-threo-p-fluorophenylserine+the amount of L-erythro-p-fluorophenylserine)]×100%.

Claims

1. A L-threonine aldolase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO:3, which is obtained by changing the 318th amino acid from Arg to Leu, the 128th amino acid from His to Asn, and the 90th amino acid from Asn to Gln of the L-threonine aldolase of the amino acid sequence SEQ ID NO:

1.

2. A gene encoding the L-threonine aldolase mutant according to claim 1.

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

4.

4. An expression cassette, vector or recombinant bacterium comprising the gene according to claim 2.

5. The method for preparing the L-threonine aldolase mutant according to claim 1, characterized in that: The method comprises the following steps: synthesizing the coding gene of the L-threonine aldolase mutant, constructing an expression vector, transforming a protein expression host bacterium, inducing protein expression and purifying the protein.

6. The method according to claim 5, characterized in that: The nucleotide sequence of the gene encoding the L-threonine aldolase mutant is shown in SEQ ID NO:

4.

7. A catalyst, characterized in that The active ingredient comprises the L-threonine aldolase mutant according to claim 1.

8. Use of the L-threonine aldolase mutant according to claim 1 or the catalyst according to claim 7 in the aldol condensation reaction of benzaldehyde or p-fluorobenzaldehyde and glycine.

9. The use according to claim 8, characterized in that: Phenylserine and its derivatives are synthesized by using the L-threonine aldolase mutant described in claim 1 or the catalyst described in claim 7, taking benzaldehyde or p-fluorobenzaldehyde as substrate, glycine as cosubstrate and 5-pyridoxal phosphate as coenzyme for catalytic reaction.

10. The use according to claim 9, characterized in that: The catalytic reaction is carried out at 15-37° C. and pH 5.0-11.0.

Images