CN114703169B - L-threonine aldolase mutant R318L/H128N and application thereof - Google Patents

Abstract

The invention relates to threonine aldolase, in particular to an L-threonine aldolase mutant R318L/H128N and application thereof. 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 318 th amino acid of the L-threonine aldolase with the amino acid sequence of SEQ ID NO. 1 from Arg to Leu and changing the 128 th amino acid from His to Asn. The mutant has 92.89% diastereoselectivity in catalyzing the reaction of synthesizing droxidopa [ L-threo- (3, 4-dihydroxyl) phenylserine ] by 3, 4-dihydroxybenzaldehyde and glycine, the yield reaches 1.26mg/mL, and the diastereoselectivity in synthesizing droxidopa is 94.5% and the yield is 1.6mg/mL when the mutant is catalyzed by whole cells expressing R318L/H128N. Whole cells have great potential for application in droxidopa biosynthesis.

Description

L-Threonine Aldolase Mutant R318L/H128N and Its Application

Technical Field

The invention relates to threonine aldolase, and in particular to an L-threonine aldolase mutant R318L/H128N and an application thereof.

Background Art

Droxidopa (L-threo-DOPS, i.e. L-threo-(3,4-dihydroxy)phenylserine) is the only drug used to treat Parkinson's-related neurogenic orthostatic hypotension (NOH) since midodrine was approved in 1996. It is used to improve gait stiffness and orthostatic dizziness caused by Parkinson's disease; to improve orthostatic hypotension, orthostatic dizziness and syncope caused by Shy-Drager syndrome or familial amyloid neuropathy; and to improve dizziness and fatigue caused by orthostatic hypotension in hemodialysis patients.

At present, the synthesis method of droxidopa includes chemical synthesis and enzyme catalysis, and its industrial production has always relied on chemical synthesis. Among them, the chemical synthesis method mainly obtains chiral droxidopa through addition reaction, esterification reaction and optical resolution. Although the operation is simple, a large amount of water, heavy metals and highly toxic hydrogen sulfide are used in the reaction process, and optical resolution will cause half of the raw materials to be wasted, the production cost is high and potential environmental pollution, which does not meet the atomic economics and environmental protection policies advocated and promoted by the country at present and in the future. The enzyme catalysis method has many advantages such as mild conditions, less water consumption (only one-tenth of the chemical method), no heavy metal pollution, no use of highly toxic chemicals, high chiral selectivity, etc., and has a good application prospect.

Threonine aldolase has a wide range of application potential. It can catalyze the synthesis of chiral pharmaceutical key intermediates β-hydroxy-α-amino acids. According to the stereospecificity of threonine aldolase on the α-carbon of the substrate threonine, the enzyme can be divided into two types: L-type and D-type. The patent application with application number 2019109657806 and invention name "Threonine aldolase, its encoding gene and application in the biosynthesis of droxidopa" discloses an L-threonine aldolase gene isolated from a black bear fecal sample, with a total length of 1032bp, encoding an L-threonine aldolase composed of 343 amino acids. The enzyme synthesizes droxidopa with a diastereoselectivity of 30% using 3-/4-hydroxy-substituted benzaldehyde and glycine as substrates. The low diastereoselectivity greatly hinders the application of the enzyme in the synthesis of droxidopa. Therefore, it is very necessary to explore highly selective threonine aldolase for the synthesis of droxidopa.

Summary of the invention

It has a significant impact on the stereoselectivity and catalytic activity of threonine aldolase

In order to develop a threonine aldolase with high selectivity, the present invention compared 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-order structure. On the premise that the site affecting the biological properties of the wild-type L-threonine aldolase was determined in the early stage as the 318th amino acid - arginine and the arginine (Arg) at this site was changed to leucine (Leu), the amino acid His128 with subunit interaction was discovered through semi-rational design and rational design, and the 128th amino acid was changed from His to Asn to obtain an enzyme mutant named L-TA R318L/H128N mutant. The coding gene (SEQ ID NO: 4) of the L-TAR318L/H128N mutant was obtained by molecular cloning technology, and the L-TA R318L/H128N 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 mutant was used as a catalyst, 3-/4-hydroxy-substituted benzaldehyde was used as a substrate, glycine was used as a cosubstrate, and 5-pyridoxal phosphate was used as a coenzyme. The enzyme catalyzed reaction was carried out under appropriate conditions and media. The results showed that the mutant synthesized droxidopa (L-threo-DOPS) with a diastereoselectivity of up to 92.89%, which was 3.09 times that of the wild type, and the yield reached 1.26 mg/mL, which has great application value in the industrial production of droxidopa.

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 SEQ ID NO: 1 from Arg to Leu and from His to Asn.

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

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

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

The vector provided by the present invention can be a cloning vector, comprising the L-TA R318L/H128N mutant gene and other elements required for plasmid replication; or it can be an expression vector, comprising the L-TA R318L/H128N 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-TA R318L/H128N mutant gene is inserted, and the pGEX-6p-2 vector is a known commercially available vector.

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 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 mutant protein is induced at 16°C.

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.

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.

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).

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 enzyme diastereoselectivity or to carry out two catalytic reactions in the same reaction system.

The use of the L-threonine aldolase mutant or the catalyst in aldol condensation reaction also falls within the protection scope of the present invention.

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 3,4-dihydroxybenzaldehyde as a substrate, glycine as a cosubstrate, and pyridoxal 5-phosphate as a coenzyme to synthesize droxidopa.

In some preferred embodiments of the present invention, the catalytic reaction is carried out at 15-37° C. and pH 6.0-11.0.

The L-TA R318L/H128N mutant of the present invention can catalyze the reversible aldol condensation reaction of 3,4-dihydroxybenzaldehyde and glycine under the reaction conditions of 15-37° C. and pH 6.0-11.0.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the synthesis of droxidopa catalyzed by L-threonine aldolase (L-TA).

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

Figure 3. HPLC spectra of the catalytic products of the L-threonine aldolase mutant R318L/H128N; the top spectrum (labeled as R318L/H128N) shows the catalytic products of the L-TAR318L/H128N mutant, the catalytic reaction is carried out at 25°C and pH 7.4 with 3,4-dihydroxybenzaldehyde as substrate, glycine as cosubstrate, and 5-pyridoxal phosphate as coenzyme; the middle spectrum (labeled as L-threo-DOPS) is the L-threo-DOPS standard; the bottom spectrum (labeled as L-DOPS) is the L-threo-DOPS and L-erythro-DOPS racemate standard. The retention time of L-erythro-DOPS is 5.5min, and the retention time of L-threo-DOPS is 6.2min.

DETAILED DESCRIPTION

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.

Main reagents and consumables:

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);

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;

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

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

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

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

L-threo-DOPS standard (CAS: 23651-95-8, product number: D4235, molecular formula: C ₉ H ₁₁ NO ₅ ), LDOPS (L-threo-DOPS and L-erythro-DOPS racemate) standard (CAS: 23651-95-8, product number: D9446), 1-heptanesulfonic acid sodium salt (CAS: 22767-50-6, molecular formula: C ₇ H ₁₅ NaO ₃ S), and 1,4-dioxane (CAS: 123-91-1, molecular formula: C ₄ H ₈ O ₂ ) were purchased from J&K Scientific;

Glycine (CAS: 56-40-6, molecular formula: C ₂ H ₅ NO ₂ , product number A502065), 3,4-dihydroxybenzaldehyde (CAS: 139-85-5, linear molecular formula: (HO) ₂ C ₆ H ₃ CHO, product number A601406), and 5-pyridoxal phosphate (PLP, CAS: 41468-25-1, molecular formula C ₈ H ₁₂ NO ₇ P, product number A610455) were purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.

LB medium

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

Preparation method: Dissolve 10g tryptone, 5g yeast extract, and 10g sodium chloride in 950mL ddH ₂ O, then adjust the pH to 7.4 with NaOH and make up to 1L with dd H ₂ O. If preparing solid culture medium, add 15g agar per liter. Sterilize with high pressure steam at 121℃ for 20min.

PBS buffer

Preparation of pH 7.4, 10mM PBS: weigh 8g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ and 0.24g KH ₂ PO ₄ , 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 with high pressure steam at 121℃ for at least 20 minutes, and store at room temperature or in a refrigerator at 4℃ for later use.

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 relevant experimental manuals or manufacturer instructions.

Example 1. Preparation of L-threonine aldolase R318L/H128N mutant

1. Design of L-threonine aldolase mutants

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.

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

MYSFKNDYSEGAHPRILETLLRTNLEQCEGYGKDTYCEEAENLIKNKLNNESIEVHFISGGTQTNLIAISAFLRPHEGVISADTGHIFVNEAGSIEATGHKVISVDVVDGKLRRDDILSVLSKFTNEHVVKPKLVYISNSTEIGTIYKKSELEELSKVCRENNLLLFMDGARLGSALSCKENDLTLEDISKLTDAFYIGGTKNGALLGEALVICNK DLQEDFRYHLKQKGAMLAKGRLLGIQFIELFKDDLFFEIGKHENDMADILRDGISRLGYEFLVDSPSNQIFPVFNNDIIRELEKNYGFNIWEKVNEEKTAIRLVTSFATKEEPCLEFIKFLSGLTNK(SEQIDNO:1)

The nucleotide sequence (1032 bp) of the wild-type L-threonine aldolase encoding gene is:

ATGTATAGTTTTAAAAATGATTATAGTGAAGGGGCACATCCTAGAATTCTTGAAACGTTGCTGAGAACAAATTTAGAACAATGTGAAGGTTACGGAAAAGATACATACTGTGAGGAAGCTGAAAACTTAATAAAAAATAAACTAAATAATGAGTCTATTGAAGTCCATTTCATATCTGGAGGTACACAAACTAACTTAATAGCAATATCTGCATTTTTAAGGCCTCATGAGGGTGTTATATCAGCAGATACAGGGCATAT ATTTGTAAATGAAGCAGGTTCAATAGAAGCACAGGACATAAGGTGATATCTGTTGATGTTGTGGATGGTAAACTAAGAAGAGACGATATACTATCAGTATTGAGTAAGTTTACTAATGAGCATGTTGTAAAACCAAAGCTTGTTTATATATCTAACTCTACTGAAATTGGAACTATATATAAAAAATCTGAATTAGAAGAGTTAAGCAAAGTTTGTAGAGAAAATAATTTATTACTATTTATGGATGGAGCAAGATTAGG ATCTGCACTTTTCTGCAAAGAAAATGATTTGACATTAGAAGATATAAGTAAATTAACTGATGCTTTTTATATCGGGGGAACTAAGAATGGAGCTCTTTTAGGAGAAGCACTTGTTATATGTAATAAAGATTTACAGGAAGATTTTAGATATCACTTAAAACAAAAAGGAGCGATGCTTGCTAAGGGAAGGTTGCTTGGAATACAGTTTATAGAATTATTTAAAGATGATTTATTTTTTGAAATAGGAAAACATGAAAATGA TATGGCTGATATATTAAGGGATGGAATAAGTAGGCTTGGATATGAATTTTTAGTAGACTCTCCATCTAATCAAATATTCCCAGTATTTAACAATGATATTATAAGAGAATTAGAGAAAAACTATGGATTTAATATATGGGAAAAAAGTAAATGAAGAGAAAACTGCAATAAGATTAGTAACATCTTTTGCAACAAAAGAAGAACCTTGTCTAGAGTTTATAAAGTTTTTAAGTGGATTAACTAATAAATAA(SEQID NO:2)

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 the 318th and 128th amino acids of wild-type L-threonine aldolase, and the corresponding nucleotide sequences are codons 952-954 and 382-384. The codons 952-954 of the wild-type L-TA gene sequence were changed from AGA to CTG, and the codons 382-384 were changed from GCT to AAT, thereby replacing the original arginine (R) at position 318 of the amino acid sequence with leucine (L) and the original histidine (H) with asparagine (N), and an enzyme mutant was obtained, named L-TA R318L/H128N 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.

2. Obtaining the L-TA R318L/H128N mutant gene

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

1. Primer design

Primers were designed for the obtained L-TA R318L/H128N 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:

Upstream primer L-TA-R318L-BamHI-F:

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

Downstream primer L-TA-R318L-Xho I-R:

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

Upstream primer L-TA-R318L/H128N-F:

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

Downstream primer L-TA-R318L/H128N-R:

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

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

2. L-TA R318L/H128N mutant gene cloning

Using the plasmid of the L-TA mutant R318L gene reported in application number 202010323801.7 and invention name: "L-threonine aldolase mutant R3318L and its application" as a template, use the upstream primer and downstream primer obtained in step 1 to amplify the L-TA R318L/H128N mutant gene according to the following PCR system and procedure.

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

The PCR program is as follows:

a. Pre-denaturation at 94℃ for 5 min;

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;

c. Extend at 72°C for 10 min.

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 mutant gene fragment was recovered using Omega Gel Extraction Kit D2500 according to the kit instructions.

3. Expression vector construction

(1) Enzyme digestion and ligation

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

Then, T4 DNA ligase was used to connect the L-TA R318L/H128N mutant gene and pGEX-6p-2 linear vector after restriction digestion: 6 μL of L-TA R318L/H128N 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-TAR318L/H128N.

(2) Conversion

Place Trans5α competent cells (full gold, CD201-01) on ice, add 10μL pGEX-6p-2/L-TA R318L/H128N after the cells thaw, and place on ice for 30 minutes. Heat shock at 42℃ 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 it on Amp+ resistance (100μg/mL) LB plate culture medium, and culture it upside down at 37℃ overnight.

(3) Positive clone screening

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.

Enzyme digestion system:

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 mutants.

4. GST fusion heterologous expression of enzyme protein

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

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 with the correct sequencing results and place them on ice for 30min. Heat shock at 42℃ for 90s, then place on ice for 2min. Add 600μL of sterile LB liquid culture medium and culture at 37℃, 150rpm shaking for 45min. 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.

(2) Protein expression and purification

a. Inoculate the protein expression strain into sterile LB liquid culture medium with a final concentration of ampicillin of 100 μg/mL at 37°C and 170 rpm.

b. When OD 600 ≈ 0.8, add IPTG at a final concentration of 0.2 mM, and induce culture overnight (12 h) at 16°C and 180 rpm. Centrifuge at 8000 rpm for 5 min to collect the cells.

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.

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.

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.

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.

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

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 enzyme solution was determined using a Bradford protein concentration determination kit (Beyotime, P0006) according to the kit instructions.

The results are shown in Figure 2. The SDS-PAGE test results show that the L-TA R318L/H128N 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. The concentration of the L-TA R318L/H128N enzyme solution was determined to be 2mg/mL.

Example 2. Synthesis of droxidopa using the L-TAR318L/H128N mutant and detection of de value

1. Synthesis of Droxidopa

In PBS buffer (pH = 7.0), 1M glycine, 60mM 3,4-dihydroxybenzaldehyde and 50μM PLP, 2.8mg purified L-TA were added, and after reacting at 37°C for 4h, the sample was ultrafiltered using an ultrafiltration tube (10kDa) to remove insoluble matter. The ultrafiltered sample was placed in a freeze dryer for freeze drying, and then an organic phase solution (90% 0.1% w/v sodium heptanesulfonate/0.1% w/v 1,4-dioxane) was added to the appropriate concentration.

2. Detection of droxidopa and calculation of de value

The sample obtained after ultrafiltration in step 1 was diluted 10 times to achieve better HPLC separation effect. The amount of droxidopa (L-threo-DOPS) and its diastereomer (L-erythro-DOPS) in the sample was detected by HPLC. HPLC instrument model: Agilent 1260. Parameter setting: chromatographic column: COSMOSIL5C18-MS4.6×150mm; wavelength 280nm, column temperature 30℃; mobile phase: 90% 0.1% (w/v, g/mL) 1-heptanesulfonic acid sodium salt/0.1% (w/v, g/mL) 1,4-dioxane (solvent is distilled water), 10% methanol; injection volume 10μL, flow rate 1mL/min. The same HPLC parameters were used to detect L-threo-DOPS standard (0.1 mg/mL, pH 7.4, 10 mM PBS) and the racemic standards of L-threo-DOPS and L-erythro-DOPS (0.01 mg/mL, pH 7.4, 10 mM PBS).

The amount of L-threo-DOPS and L-erythro-DOPS in the sample was determined based on the peak area. The de value (diastereoisomeric excess) was then determined based on the following equation:

[(Amount of L-threo-DOPS - Amount of L-erythro-DOPS)/(Amount of L-threo-DOPS + Amount of L-erythro-DOPS)] × 100%.

The HPLC results are shown in Figure 3, where the top spectrum (labeled as R318L/H128N) is the catalytic product of the L-TAR318L/H128N mutant; the middle spectrum (labeled as L-threo-DOPS) is the L-threo-DOPS standard; the bottom spectrum (labeled as L-DOPS) is the standard of L-threo-DOPS and L-erythro-DOPS racemates. The retention time of L-erythro-DOPS is 5.5min, and the retention time of L-threo-DOPS is 6.2min. It is calculated that the L-TAR318L/H128N mutant has a diastereoselectivity (de value) of 84.9% in the reaction of catalyzing 3,4-dihydroxybenzaldehyde and glycine to synthesize droxidopa (L-threo-DOPS).

Claims (10)

1. An L-threonine aldolase mutant, characterized in that its amino acid sequence is as shown in SEQ ID NO: 3, and it is an L-threonine aldolase whose amino acid sequence is SEQ ID NO: 1. The 318th amino acid was changed from Arg to Leu and the 128th amino acid was changed from His to Asn. 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 containing the gene of claim 2. 5. The preparation method of the L-threonine aldolase mutant according to claim 1, characterized in that it includes the following steps: synthesizing the coding gene of the L-threonine aldolase mutant and constructing an expression vector , transform protein expression host bacteria, induce protein expression and purify. 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 its active ingredient contains the L-threonine aldolase mutant according to claim 1. 8. Application of the L-threonine aldolase mutant of claim 1 or the catalyst of claim 7 in the aldol condensation reaction of 3,4-dihydroxybenzaldehyde and glycine. 9. Application according to claim 8, characterized in that, using the L-threonine aldolase mutant according to claim 1 or the catalyst according to claim 7, 3,4-dihydroxybenzaldehyde is used. It is the substrate, glycine is the co-substrate, and pyridoxal-5-phosphate is the coenzyme to catalyze the reaction to synthesize droxidopa. 10. The application according to claim 9, characterized in that the catalytic reaction is carried out at 15-37°C and pH 6.0-11.0.