CN111849951B - L-threonine aldolase mutant R318M and its application - Google Patents

Abstract

The invention relates to threonine aldolase, in particular to an L-threonine aldolase mutant R318M and application thereof. The amino acid sequence of the L-threonine aldolase mutant is shown as SEQ ID NO. 3, and the amino acid at the 318 th site of the L-threonine aldolase with the amino acid sequence of SEQ ID NO. 1 is obtained by changing Arg into Met. The mutant has 77.1% diastereoselectivity (de) in the reaction of catalyzing 3, 4-dihydroxybenzaldehyde and glycine to synthesize droxidopa [ L-threo- (3, 4-dihydroxy) phenylserine ], is more than 2.5 times of that of a wild type, and has great application potential in industrial production of biosynthesis of droxidopa.

Description

L-threonine aldolase mutant R318M and its application

technical field

The present invention relates to threonine aldolase, in particular to an L-threonine aldolase mutant R318M and its application.

Background technique

Droxidopa (L-threo-DOPS, L-threo-3,4-dihydroxyphenylserine) is a β-hydroxyamino acid with two chiral centers and is an anti-Parkinson's disease drug approved by the FDA in 2014 for improving the Stiff gait and orthostatic dizziness due to Parkinson's disease; improvement in orthostatic hypotension, orthostatic dizziness and syncope due to Shy-Drager syndrome or familial amyloid neuropathy; improvement in patients with hemodialysis due to orthostatic hypotension Dizziness and fatigue caused.

At present, the synthetic methods of droxidopa mainly include chemical synthesis and enzymatic catalysis. Among them, the chemical synthesis method mainly obtains chiral droxidopa through addition reaction, esterification reaction and chemical 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 there are a large number of optical isomers as by-products, which do not conform to 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, and high chiral selection, and has a good application prospect.

The formation of asymmetric CC bonds is one of the most challenging reactions in synthetic organic chemistry. Enzymatic aldol additions offer enormous opportunities to synthesize compounds with asymmetric CC bonds, especially various chiral β-hydroxyamino acids. Threonine aldolase has a very wide range of potential applications, it can catalyze the synthesis of chiral pharmaceutical key intermediate β-hydroxy-α-amino acid. According to the stereospecificity of threonine aldolase to the substrate threonine α carbon, the enzyme can be divided into two types: L-type and D-type. L-threonine aldolase (L-TA) is a pyrrole aldehyde 5'-phosphate (PLP)-dependent aldolase that catalyzes various β-hydroxy-α-L derived from glycine and various aldehydes in one step - Optical synthesis of amino acids. For alkylaldehydes, L-TA always provides excellent stereoselectivity. However, for β-hydroxy- _α -L-amino acids synthesized from aromatic aldehydes, L-TA is highly stereoselective (>99% diastereomeric excess, de) at Cα, while at _Cβ Shows moderate to low stereoselectivity (about 10-45% de), especially for those aromatic groups with electron donating groups such as L-threo-DOPS.

The patent application with the application number of 2019109657806 and the invention titled "Threonine aldolase, its encoding gene and its application in the biosynthesis of droxidopa" discloses an L-threonine isolated from black bear fecal samples Aldolase gene, full-length 1032bp, encodes L-threonine aldolase composed of 343 amino acids. The diastereoselectivity of the enzyme to synthesize droxidopa (L-threo-DOPS) with 3-/4-hydroxyl-substituted benzaldehyde and glycine as substrates was 30%. The low diastereoselectivity greatly hinders the application of this enzyme in the synthesis of L-threo-DOPS. Therefore, it is very necessary to explore threonine aldolase with high selectivity for the synthesis of droxidopa.

SUMMARY OF THE INVENTION

In order to develop a threonine aldolase with high selectivity, the present invention compared the wild-type L-threonine aldolase (amino acid sequence) isolated from black bear feces samples from the primary structure to the higher structure. As shown in SEQ ID NO: 1) and the homologous enzyme protein, it is determined that the site that affects the properties of wild-type L-threonine aldolase is the 318th amino acid - arginine. Then, the arginine (Arg) at this site was changed to methionine (Met) by codon substitution to obtain an enzyme mutant, named L-TA R318M mutant. The coding gene (SEQ ID NO: 4) of the L-TA R318M mutant was obtained by molecular cloning technology, and the L-TA was obtained by constructing the GST fusion expression vector of the mutant gene and introducing it into the genetically engineered bacteria E.coliBL21 to induce expression. R318M mutant enzyme protein. Using L-TA R318M mutant as catalyst, 3-/4-hydroxyl-substituted benzaldehyde as substrate, glycine as cosubstrate, and pyridoxal 5-phosphate as coenzyme, enzymatic catalysis was carried out under appropriate conditions and media. The results showed that the diastereoselectivity of the mutant to synthesize droxidopa (L-threo-DOPS) reached 77.1%, which was more than 2.5 times that of the wild-type enzyme. huge application value.

The present invention provides an L-threonine aldolase mutant, characterized in that its amino acid sequence is shown in SEQ ID NO: 3, and it is the L-threonine aldolase whose amino acid sequence is SEQ ID NO: 1. Amino acid 318 was changed from Arg to Met.

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.

Expression cassettes, vectors or recombinant bacteria containing the genes also belong to the protection scope of the present invention.

The vector provided by the present invention can be a cloning vector, including the L-TA R318M mutant gene and other elements required for plasmid replication; it can also be an expression vector, including the L-TA R318M mutant gene and other elements capable of successfully expressing the protein element. In some embodiments of the present invention, the expression vector is the pGEX-6p-2 vector into which the L-TA R318M mutant gene is inserted, and the pGEX-6p-2 vector is a known commercial vector.

The recombinant bacteria provided by the present invention can be a recombinant bacteria containing a cloning vector, such as E.coli DH5α, by culturing the recombinant bacteria to replicate the L-TA R318M mutant gene; it can also be a recombinant bacteria containing an expression vector. Cultivate the recombinant bacteria under conditions and express the protein, for example, add an appropriate amount of IPTG to the culture medium of the recombinant bacteria to induce the expression of the L-TA R318M mutant protein at 16°C.

The present invention also provides a method for preparing the L-threonine aldolase mutant, comprising the steps of: synthesizing the encoding gene of the L-threonine aldolase mutant, constructing an expression vector, and transforming a protein expression host bacteria , induce protein expression and purify.

In a preferred embodiment of the preparation method of the present invention, the nucleotide sequence of the encoding gene of 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 pGEX-6p-2 vector, and the protein expression host strain 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, so as to improve the diastereoselectivity of the enzyme or to carry out two catalytic reactions successively in the same reaction system.

The application of the L-threonine aldolase mutant or the catalyst in the aldol condensation reaction also belongs to 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, with 3,4-dihydroxybenzaldehyde as a substrate, glycine as a co-substrate, and 5-phosphate Pyridoxal is used as a coenzyme to catalyze the reaction 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 R318M 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.

Description of drawings

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

Figure 2. The agarose gel electrophoresis results of the L-TA R318M mutant gene constructed by PCR; wherein, M is DNAMarker; R318M is the PCR product (L-TA R318M mutant gene).

Figure 3. The identification results of double-enzyme digestion of recombinant plasmid pGEX-6p-2/L-TA R318M; wherein, M is DNA Marker; R318M is the digestion product.

Figure 4. SDS-PAGE electropherogram of L-threonine aldolase mutant R318M; wherein, M is a protein molecular weight marker (Marker); R318M is a mutant protein with a molecular weight of about 38.7KD.

Figure 5. HPLC profile of the catalytic product of the L-threonine aldolase mutant R318M; wherein the top spectrum (labeled as R318M) shows the catalytic product of the L-TA R318M mutant, the catalytic reaction is 3, 4-Dihydroxybenzaldehyde as substrate, glycine as cosubstrate, and pyridoxal 5-phosphate as coenzyme, at 25°C, pH 7.4; the middle spectrum (labeled as L-threo-DOPS) is the L-threo-DOPS standard; the bottom spectrum (labeled L-DOPS) is the L-threo-DOPS and L-erythro-DOPS racemate standard. The retention time of L-erythro-DOPS was 5.5 min, and the retention time of L-threo-DOPS was 6.2 min.

Detailed ways

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

Main reagents and consumables:

PrimeSTAR Max Premix (2×) (Cat. No. R045A), BamH I Restriction Enzyme (Cat. No. 1010S), Xho I Restriction Enzyme (Cat. No. 1094S), T4 DNA Ligase (Cat. No. D2011A) and 10×T4 Ligase buffer, All purchased from Bao Biotechnology Co., Ltd. (Dalian);

The pGEX-6p-2 plasmid is a known E. coli expression vector, alias pGEX6P2, pGEX 6P 2, the vector size is 4985bp, Tac promoter, vector tag N-GST, vector resistance Ampicillin (ampicillin), purchased from Shanghai Sheng Engineering Biotechnology Co., Ltd., the laboratory is also preserved;

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, Cat. No. 10223836;

PreScission Protease, purchased from GenScript, Item No. Z02799-100;

Bradford Protein Concentration Assay Kit, purchased from Beyotime Company, Item No. P0006;

L-threo-DOPS standard (CAS: 23651-95-8, product number: D4235, molecular formula: C ₉ H ₁₁ NO ₅ ), L-DOPS (L-threo-DOPS and L-erythro-DOPS racemates ) standard product (CAS: 23651-95-8, product number: D9446), 1-heptanesulfonic acid sodium salt (CAS: 22767-50-6, molecular formula: C7H15NaO3S), 1,4-dioxane (CAS: 123-91-1, molecular formula: C ₄ H ₈ O ₂ ) were purchased from Bailingwei Technology Company;

Glycine (CAS: 56-40-6, molecular formula: C ₂ H ₅ NO ₂ , Cat. No. A502065), 3,4-dihydroxybenzaldehyde (CAS: 139-85-5, linear formula: (HO) ₂ C ₆ H ₃ CHO, product number A601406), pyridoxal 5-phosphate (English name PLP, CAS: 41468-25-1, molecular formula C ₈ H ₁₂ NO ₇ P, product number A610455), all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. company.

LB medium

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

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 make up to 1L with ddH2O. If preparing solid medium, add 15 g of agar per liter. Autoclave at 121°C for 20min.

PBS buffer

The preparation method 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 800 mL of distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water Make up to 1L. Sterilize by autoclaving at 121°C for at least 20 minutes, and store at room temperature or in a refrigerator at 4°C for later use.

Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the field, which can be obtained commercially or prepared according to conventional methods in the field, 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 may be made to relevant experimental manuals or manufacturer's instructions.

Example 1. Preparation of L-threonine aldolase R318M mutant

1. Design of L-threonine aldolase mutants

The wild-type L-threonine aldolase gene (L-TA gene, SEQ ID NO: 2) was isolated in our laboratory from the fecal samples of healthy black bears in the Sichuan black bear conservation and incubation base. The full length of the open reading frame is 1032bp, and the encoded L-threonine aldolase consists of 343 amino acids. The isolation and cloning process of the gene is recorded in the text of the patent application with the application number of 2019109657806, the publication number of CN110592058A, and the title of the invention is "Threonine aldolase, its encoding gene and its application in the biosynthesis of droxidopa" , the entire contents of this patent application are incorporated herein by reference.

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

MYSFKNDYSEGAHPRILETLLRTNLEQCEGYGKDTYCEEAENLIKNKLNNESIEVHFISGGTQTNLIAISAFLRPHEGVISADTGHIFVNEAGSIEATGHKVISVDVVDGKLRRDDILSVLSKFTNEHVVKPKLVYISNSTEIGTIYKKSELEELSKVCRENNLLLFMDGARLGSALSCKENDLTLEDISKLTDAFYIGGTKNGALLGEALVICNKDLQEDFRYHLKQKGAMLAKGRLLGIQFIELFKDDLFFEIGKHENDMADILRDGISRLGYEFLVDSPSNQIFPVFNNDIIRELEKNYGFNIWEKVNEEKTAI R LVTSFATKEEPCLEFIKFLSGLTNK(SEQ ID NO:1)

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

AGA TTAGTAACATCTTTTGCAACAAAAGAAGAACCTTGTCTAGAGTTTATAAAGTTTTTAAGTGGATTAACTAATAAATAA (SEQ ID NO: 2)

We compared the similarities and differences between the wild-type L-threonine aldolase and the homologous enzyme protein from the multi-angle multi-layer system from the primary structure to the higher structure, and determined that the site affecting the enzymatic properties was the wild-type L-threonine aldolase The 318th amino acid of acid aldolase is arginine (R), and its corresponding nucleotide sequence is the 952nd-954th codon. The codons 952-954 of the wild-type L-TA gene sequence were changed from AGA to ATG, and the 318th position of the amino acid sequence was replaced by the original arginine (R) with methionine (M), resulting in The enzyme mutant, named L-TA R318M mutant, its 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. Acquisition of L-TA R318M mutant gene

The L-TA R318M mutant gene can be obtained by a total gene synthesis method or a molecular cloning method. In this experiment, the mutant gene was obtained by PCR method.

1. Primer design

Design primers for the obtained L-TA R318M mutant gene sequence (SEQ ID NO: 4), add restriction site BamH I (GGATCC) to the 5' end primer, and add restriction enzyme site Xho to the 3' end primer I(CTCGAG). The nucleotide sequences of the primers are as follows:

The upstream primer is: L-TA-R318M-BamHI-F

5'-CGC GGATCC ATGTATAGTTTTAAAAATGATTATA-3' (SEQ ID NO: 5),

The downstream primer is: L-TA-R318M-XhoI-R

5'-CCG CTCGAG TTATTTATTAGTTAATCCACTTAAAAACTTTATAAACTCTAGACAAGGTTCTTCTTTTGTTGCAAAAGATGTTACTAACATTATTG-3' (SEQ ID NO: 6).

Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to synthesize the above primers.

2. L-TA R318M mutant gene cloning

Using the plasmid containing the wild-type L-TA gene as a template, and using the upstream and downstream primers obtained in step 1, the L-TA R318M mutant gene was amplified according to the following PCR system and procedure. The preparation method of the plasmid containing the wild-type L-TA gene is recorded in the application number 2019109657806, the publication number is CN110592058A, and the name of the invention is "threonine aldolase, its encoding gene and doxidopa biosynthesis. In the text of the patent application "application", it is described as plasmid pGEX-6p-2/L-TA Sz-1-2 in its Example 3, and the entire content of the patent application is incorporated herein by reference.

PCR system: PrimeSTAR Max Premix (2×) 25 μL, plasmid template 0.5 μL, upstream primer L-TA-R318M-BamHI-F (10 μM) 2 μL, downstream primer L-TA-R318M-XhoI-R (10 μM) 2 μL, 0.1 % ( _g /ml) BSA was 3 [mu]L, supplemented with ddH2O to make the PCR system 50 [mu]L.

The PCR procedure is as follows:

a. Pre-denaturation at 94°C for 5min;

b. Denaturation at 98°C for 10sec, annealing at 48°C for 10sec, extension at 72°C for 10sec; 40 cycles;

c. Extend at 72°C for 10min.

The PCR amplification products were detected by 1.2% agarose gel electrophoresis, and the results were shown in Figure 2, and a target band with a size of about 1000 bp was obtained. The target band was cut under UV light, and the L-TA R318M mutant gene fragment was recovered using the Omega Gel Extraction Kit D2500 according to the kit instructions.

3. Construction of expression vector

(1) Enzyme cleavage and ligation

The L-TA R318M mutant gene and pGEX-6p-2 vector were double digested with BamH I and Xho I restriction enzymes, respectively. Enzyme digestion system (gene): 35 μL of L-TA R318M mutant gene, 3 μL of BamH I enzyme, 3 μL of Xho I enzyme, 6 μL of 10×K buffer, supplemented with sterile double-distilled water to make the system 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, supplemented with sterile double-distilled water to make the system 10 μL. Digestion conditions: 37°C for 3h.

Then use T4 DNA ligase to ligase the L-TA R318M mutant gene and pGEX-6p-2 linear vector:

After ligation at 16°C overnight, the ligation product pGEX-6p-2/L-TA R318M was obtained.

(2) Conversion

Trans5α competent cells (full gold, CD201-01) were placed on ice, 10 μL of pGEX-6p-2/L-TA R318M was added after the cells were thawed, and placed on ice for 30 min. Heat shock at 42°C for 90s, then place on ice for 2min. Add 600 μL of sterile LB liquid medium, and cultivate at 37° C. with a shaker at 150 rpm for 45 min. Pipette 200 μL of the cultured bacterial liquid, spread it on Amp ⁺ resistant (100 μg/mL) LB plate medium, and invert at 37°C overnight.

(3) Screening of positive clones

Pick a single colony on the LB plate, inoculate it in Amp ⁺ resistant (100 μg/mL) LB liquid medium, culture at 37°C, shaker at 220 rpm to OD600≈1.0, and centrifuge at 8000 rpm for 5 min to collect bacteria for plasmid extraction. The plasmid was extracted using OMEGAPlasmid Mini Kit I (Catalog No.: D6943) according to the instructions, and the plasmid was identified by double restriction digestion.

Enzyme cleavage system:

Digestion conditions: 37°C for 3h. Use 1.2% agarose gel electrophoresis to detect the digested product, select the correct recombinant plasmid identified by double enzyme digestion and send it to TAKARA (Dalian, China) for sequencing. The correct recombinant plasmid is used as the expression vector of the L-TAR318M mutant.

4. GST fusion heterologous expression of enzyme protein

(1) E.coli BL21(DE3) cells were transformed with plasmids

Remove E. coli BL21(DE3) competent cells from -80°C and place on ice. After the cells were thawed, 10 μL of the expression vector pGEX-6p-2/L-TA R318M with correct sequencing results was added and placed on ice for 30 min. Heat shock at 42°C for 90s, then place on ice for 2min. Add 600 μL of sterile LB liquid medium, and cultivate at 37° C. with a shaker at 150 rpm for 45 min. Pipette 200 μL of the cultured bacterial liquid, spread it on Amp ⁺ resistant (100 μg/mL) LB plate medium, and invert at 37°C overnight. Pick a single colony on the LB plate, carry out 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 medium, the final concentration of ampicillin is 50 μg/mL, and cultivate at 37° C. and 180 rpm.

b. When OD600≈0.8, add IPTG with a final concentration of 0.2 mM, induce and culture at 16° C. and 180 rpm overnight (12 h). The cells were collected by centrifugation at 8000 rpm for 5 min.

c. Add 30 mL of lysis buffer (pH 7.4, 10 mM PBS) to the 1 L culture system to resuspend the bacteria, and sterilize the bacteria by sonication at 4° C. until clear. The broken bacterial liquid was evenly divided into 50mL sterile centrifuge tubes pre-cooled at 4°C, and centrifuged at 12000rpm for 20min at 4°C. After the centrifugation was completed, the supernatant was transferred to a 50mL sterile centrifuge tube pre-cooled at 4°C with a precision pipette. middle.

d. Pack Glutathione Sepharose 4B filler (GE Healthcare) into a chromatography column (GE Healthcare), and the ratio of filler used is 5mL filler per liter of culture system. Absolute ethanol was removed by washing with 3 column volumes of 10 mM sterile PBS, pH 7.4, pre-chilled at 4°C. The supernatant obtained in step c was combined with Glutathione Sepharose 4B for 2 hours at 4°C. Gently invert the suspension vertically.

e. After the binding is completed, centrifuge at 500 rpm for 5 min to precipitate the filler. The filler was washed with 4°C pre-cooled pH7.4, 10mM sterile PBS for 3-5 column volumes to remove impurity proteins.

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

g. After the enzyme digestion is completed, the supernatant is released from the chromatography column to obtain L-TA R318M enzyme solution.

h. The L-TA R318M enzyme solution was detected by SDS-PAGE to identify its molecular weight. The concentration of L-TA R318M enzyme solution was measured using Bradford protein concentration assay kit (Beyotime, P0006) according to the kit instructions.

The results are shown in Figure 4. The results of SDS-PAGE showed that the soluble expression of the L-TA R318M mutant was successful, the protein molecular weight was about 38.7KD, and the enzyme protein was very pure, showing a single band. The concentration of L-TA R318M enzyme solution was determined to be 2 mg/mL.

Example 2. Synthesis of droxidopa using L-TA R318M mutant and detection of de value

1. Synthesis of Droxidopa

The substrate solution was prepared in 10 mM PBS, pH 7.4, containing 1 M glycine, 60 mM 3,4-dihydroxybenzaldehyde and 50 [mu]M pyridoxal 5-phosphate. The L-TA R318M enzyme 20U prepared in Example 1 was added to each milliliter of the substrate solution, mixed well, placed at 25°C for reaction for 2h, and then ultrafiltered (10kDa, 4000×g, 30min) to obtain the sample.

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 samples was determined by HPLC. HPLC instrument model: Agilent 1260. Parameter setting: Column: COSMOSIL 5C18-MS 4.6×150mm; wavelength 280nm, column temperature 30°C; mobile phase: 90% 0.1% (w/v, g/mL) 1-heptanesulfonic acid sodium salt/0.1% ( w/v, g/mL) 1,4-dioxane (the solvent is distilled water), 10% methanol; the injection volume is 10 μL, and the flow rate is 1 mL/min. L-threo-DOPS standard (0.1 mg/mL, pH 7.4, 10 mM PBS), and L-threo-DOPS and L-erythro-DOPS racemic standard (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, percent 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 5, where the top spectrum (labeled as R318M) is the catalytic product of the L-TA R318M mutant; the middle spectrum (labeled as L-threo-DOPS) is L-threo-DOPS Standard; the bottom spectrum (labeled L-DOPS) is the standard for the L-threo-DOPS and L-erythro-DOPS racemates. The retention time of L-erythro-DOPS was 5.5 min, and the retention time of L-threo-DOPS was 6.2 min. The L-TA R318M mutant was calculated to have a diastereoselectivity (de value) of 77.1% in catalyzing the synthesis of droxidopa (L-threo-DOPS) from 3,4-dihydroxybenzaldehyde and glycine.

sequence listing

<110> Chongqing University

<120> L-threonine aldolase mutant R318M and its application

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Met Tyr Ser Phe Lys Asn Asp Tyr Ser Glu Gly Ala His Pro Arg Ile

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Lys Asp Thr Tyr Cys Glu Glu Ala Glu Asn Leu Ile Lys Asn Lys Leu

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Ser Ala Asp Thr Gly His Ile Phe Val Asn Glu Ala Gly Ser Ile Glu

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Arg Arg Asp Asp Ile Leu Ser Val Leu Ser Lys Phe Thr Asn Glu His

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Glu Asn Asn Leu Leu Leu Phe Met Asp Gly Ala Arg Leu Gly Ser Ala

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Leu Ser Cys Lys Glu Asn Asp Leu Thr Leu Glu Asp Ile Ser Lys Leu

180 185 190

Thr Asp Ala Phe Tyr Ile Gly Gly Thr Lys Asn Gly Ala Leu Leu Gly

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Glu Ala Leu Val Ile Cys Asn Lys Asp Leu Gln Glu Asp Phe Arg Tyr

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atgtatagtt ttaaaaatga ttatagtgaa ggggcacatc ctagaattct tgaaacgttg 60

ctgagaacaa atttagaaca atgtgaaggt tacggaaaag atacatactg tgaggaagct 120

gaaaacttaa taaaaaataa actaaataat gagtctattg aagtccattt catatctgga 180

ggtacacaaa ctaacttaat agcaatatct gcatttttaa ggcctcatga gggtgttata 240

tcagcagata cagggcatat atttgtaaat gaagcaggtt caatagaagc aacaggacat 300

aaggtgatat ctgttgatgt tgtggatggt aaactaagaa gagacgatat actatcagta 360

ttgagtaagt ttactaatga gcatgttgta aaaccaaagc ttgtttatat atctaactct 420

actgaaattg gaactatata taaaaaatct gaattagaag agttaagcaa agtttgtaga 480

gaaaataatt tattactatt tatggatgga gcaagattag gatctgcact ttcttgcaaa 540

gaaaatgatt tgacattaga agatataagt aaattaactg atgcttttta tatcggggga 600

actaagaatg gagctctttt aggagaagca cttgttatat gtaataaaga tttacaggaa 660

gattttagat atcacttaaa acaaaaagga gcgatgcttg ctaagggaag gttgcttgga 720

atacagttta tagaattatt taaagatgat ttattttttg aaataggaaa acatgaaaat 780

gatatggctg atatattaag ggatggaata agtaggcttg gatatgaatt tttagtagac 840

tctccatcta atcaaatatt cccagtattt aacaatgata ttataagaga attagagaaa 900

aactatggat ttaatatatg ggaaaaagta aatgaagaga aaactgcaat aagattagta 960

acatcttttg caacaaaaga agaaccttgt ctagagttta taaagtttttt aagtggatta 1020

actaataaat aa 1032

<210> 3

<211> 343

<212> PRT

<213> Artificial Sequence

<400> 3

Met Tyr Ser Phe Lys Asn Asp Tyr Ser Glu Gly Ala His Pro Arg Ile

1 5 10 15

Leu Glu Thr Leu Leu Arg Thr Asn Leu Glu Gln Cys Glu Gly Tyr Gly

20 25 30

Lys Asp Thr Tyr Cys Glu Glu Ala Glu Asn Leu Ile Lys Asn Lys Leu

35 40 45

Asn Asn Glu Ser Ile Glu Val His Phe Ile Ser Gly Gly Thr Gln Thr

50 55 60

Asn Leu Ile Ala Ile Ser Ala Phe Leu Arg Pro His Glu Gly Val Ile

65 70 75 80

Ser Ala Asp Thr Gly His Ile Phe Val Asn Glu Ala Gly Ser Ile Glu

85 90 95

Ala Thr Gly His Lys Val Ile Ser Val Asp Val Val Asp Gly Lys Leu

100 105 110

Arg Arg Asp Asp Ile Leu Ser Val Leu Ser Lys Phe Thr Asn Glu His

115 120 125

Val Val Lys Pro Lys Leu Val Tyr Ile Ser Asn Ser Thr Glu Ile Gly

130 135 140

Thr Ile Tyr Lys Lys Ser Glu Leu Glu Glu Leu Ser Lys Val Cys Arg

145 150 155 160

Glu Asn Asn Leu Leu Leu Phe Met Asp Gly Ala Arg Leu Gly Ser Ala

165 170 175

Leu Ser Cys Lys Glu Asn Asp Leu Thr Leu Glu Asp Ile Ser Lys Leu

180 185 190

Thr Asp Ala Phe Tyr Ile Gly Gly Thr Lys Asn Gly Ala Leu Leu Gly

195 200 205

Glu Ala Leu Val Ile Cys Asn Lys Asp Leu Gln Glu Asp Phe Arg Tyr

210 215 220

His Leu Lys Gln Lys Gly Ala Met Leu Ala Lys Gly Arg Leu Leu Gly

225 230 235 240

Ile Gln Phe Ile Glu Leu Phe Lys Asp Asp Leu Phe Phe Glu Ile Gly

245 250 255

Lys His Glu Asn Asp Met Ala Asp Ile Leu Arg Asp Gly Ile Ser Arg

260 265 270

Leu Gly Tyr Glu Phe Leu Val Asp Ser Pro Ser Asn Gln Ile Phe Pro

275 280 285

Val Phe Asn Asn Asp Ile Ile Arg Glu Leu Glu Lys Asn Tyr Gly Phe

290 295 300

Asn Ile Trp Glu Lys Val Asn Glu Glu Lys Thr Ala Ile Met Leu Val

305 310 315 320

Thr Ser Phe Ala Thr Lys Glu Glu Pro Cys Leu Glu Phe Ile Lys Phe

325 330 335

Leu Ser Gly Leu Thr Asn Lys

340

<210> 4

<211> 1032

<212> DNA

<213> Artificial Sequence

<400> 4

atgtatagtt ttaaaaatga ttatagtgaa ggggcacatc ctagaattct tgaaacgttg 60

ctgagaacaa atttagaaca atgtgaaggt tacggaaaag atacatactg tgaggaagct 120

gaaaacttaa taaaaaataa actaaataat gagtctattg aagtccattt catatctgga 180

ggtacacaaa ctaacttaat agcaatatct gcatttttaa ggcctcatga gggtgttata 240

tcagcagata cagggcatat atttgtaaat gaagcaggtt caatagaagc aacaggacat 300

aaggtgatat ctgttgatgt tgtggatggt aaactaagaa gagacgatat actatcagta 360

ttgagtaagt ttactaatga gcatgttgta aaaccaaagc ttgtttatat atctaactct 420

actgaaattg gaactatata taaaaaatct gaattagaag agttaagcaa agtttgtaga 480

gaaaataatt tattactatt tatggatgga gcaagattag gatctgcact ttcttgcaaa 540

gaaaatgatt tgacattaga agatataagt aaattaactg atgcttttta tatcggggga 600

actaagaatg gagctctttt aggagaagca cttgttatat gtaataaaga tttacaggaa 660

gattttagat atcacttaaa acaaaaagga gcgatgcttg ctaagggaag gttgcttgga 720

atacagttta tagaattatt taaagatgat ttattttttg aaataggaaa acatgaaaat 780

gatatggctg atatattaag ggatggaata agtaggcttg gatatgaatt tttagtagac 840

tctccatcta atcaaatatt cccagtattt aacaatgata ttataagaga attagagaaa 900

aactatggat ttaatatatg ggaaaaagta aatgaagaga aaactgcaat aatgttagta 960

acatcttttg caacaaaaga agaaccttgt ctagagttta taaagtttttt aagtggatta 1020

actaataaat aa 1032

<210> 5

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 5

cgcggatcca tgtatagttt taaaaatgat tata 34

<210> 6

<211> 95

<212> DNA

<213> Artificial Sequence

<400> 6

ccgctcgagt tatttattag ttaatccact taaaaacttt ataaactcta gacaaggttc 60

ttcttttgtt gcaaaagatg ttactaacat tattg 95

Claims (10)

1. an L-threonine aldolase mutant, is characterized in that, its amino acid sequence is as shown in SEQ ID NO:3, is that the aminoacid sequence is the L-threonine aldolase of SEQ ID NO:1 Amino acid 318 was changed from Arg to Met. 2. A gene encoding the L-threonine aldolase mutant of claim 1. 3. The gene according to claim 2, wherein the nucleotide sequence is as shown in SEQ ID NO:4. 4. An expression cassette, vector or recombinant bacteria comprising the gene of claim 2. 5. the preparation method of L-threonine aldolase mutant according to claim 1, is characterized in that, comprises the steps: synthesizing the coding gene of described L-threonine aldolase mutant, constructing expression vector , transform the host bacteria for protein expression, induce protein expression and purify. 6 . The method according to claim 5 , wherein the nucleotide sequence of the encoding gene of the L-threonine aldolase mutant is shown in SEQ ID NO: 4. 7 . 7 . A catalyst, wherein the active ingredient comprises the L-threonine aldolase mutant of claim 1 . 8 . 8. Use of the L-threonine aldolase mutant of claim 1 or the catalyst of claim 7 in an aldol condensation reaction. 9. The 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, with 3,4-dihydroxybenzaldehyde As a substrate, glycine as a cosubstrate, and pyridoxal 5-phosphate as a coenzyme to catalyze the reaction to synthesize droxidopa. 10. The application according to claim 9, wherein the catalytic reaction is carried out under the conditions of 15-37°C and pH 6.0-11.0.

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