CN111849951B

CN111849951B - L-threonine aldolase mutant R318M and application thereof

Info

Publication number: CN111849951B
Application number: CN202010324327.XA
Authority: CN
Inventors: 王伯初; 赵文艳; 潘银平; 杨碧玲; 祝连彩; 戚娜
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2022-09-20
Anticipated expiration: 2040-04-22
Also published as: CN111849951A

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 application thereof

Technical Field

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

Background

Droxidopa (L-threo-DOPS, L-threo-3,4-dihydroxyphenylserine) has beta-hydroxy amino acid with two chiral centers, is an antiparkinsonian drug approved by FDA in 2014, and is used for improving gait rigidity and erect dizziness caused by Parkinson disease; ameliorating orthostatic hypotension, orthostatic dizziness and fainting caused by Shy-Drager syndrome or familial amyloid neuropathy; improve dizziness and hypodynamia of hemodialysis patients caused by orthostatic hypotension.

At present, the synthetic method of droxidopa mainly comprises chemical synthesis and enzymatic catalysis. The chemical synthesis method is mainly used for obtaining chiral droxidopa through addition reaction, esterification reaction and chemical resolution, although the operation is simple, a large amount of water, heavy metals and virulent hydrogen sulfide are used in the reaction process, and a large amount of optical isomers are used as byproducts, so that the method does not meet the atomic economy and environmental protection policies advocated and promoted by the current and future countries. The enzyme catalysis method has the advantages of mild condition, less water consumption (only one tenth of that of the chemical method), no heavy metal pollution, no use of highly toxic chemicals, high chiral selection and the like, and has good application prospect.

The formation of asymmetric C-C bonds is one of the most challenging reactions in synthetic organic chemistry. The enzymatic aldol addition reaction provides a great opportunity for the synthesis of compounds with asymmetric C-C bonds, in particular various chiral beta-hydroxyamino acids. Threonine aldolase has very wide application potential, and can catalyze and synthesize chiral pharmaceutical key intermediate beta-hydroxy-alpha-amino acid. Threonine aldolase can be classified into two types, L-type and D-type, according to its stereospecificity at the alpha carbon of threonine as a substrate. L-threonine aldolase (L-TA) is a pyrrole aldehyde 5' -phosphate (PLP) -dependent aldolase that can catalyze the optical synthesis of a variety of β -hydroxy- α -L-amino acids derived from glycine and a variety of aldehydes in one step. For alkylaldehydes, L-TA can always provide excellent stereoselectivity. However, for the aromatic aldehyde synthesized beta-hydroxy-alpha-L-amino acids, L-TA is at C _α Has high stereoselectivity of (>99% diastereomeric excess, de), and in C _β And exhibits moderate to low stereoselectivity (about 10-45% de), particularly for those aromatic groups bearing electron donating groups, such as L-threo-DOPS.

The patent application with the title of 'threonine aldolase, coding gene and application in droxidopa biosynthesis' with the application number of 2019109657806 discloses an L-threonine aldolase gene separated from a black bear stool sample, with the total length of 1032bp, coding for an L-threonine aldolase consisting of 343 amino acids. The diastereoselectivity of the enzyme for synthesizing droxidopa (L-threo-DOPS) by using benzaldehyde and glycine substituted by hydroxyl at the 3-/4-position as substrates is 30%. The use of this enzyme in the synthesis of L-threo-DOPS is largely hampered by the low diastereoselectivity. Therefore, it is very necessary to develop threonine aldolase with high selectivity for the synthesis of droxidopa.

Disclosure of Invention

In order to develop threonine aldolase with high selectivity, the invention compares the dissimilarity of wild-type L-threonine aldolase (amino acid sequence is shown as SEQ ID NO: 1) separated from a black bear feces sample and homologous enzyme protein from a primary structure to a high-level structure multi-layer system, and determines that the site influencing the optical property of the wild-type L-threonine aldolase is arginine at the 318 th amino acid. The arginine (Arg) at this site was then changed to methionine (Met) by codon substitution, resulting in an enzyme mutant designated L-TA R318M mutant. The encoding gene (SEQ ID NO:4) of the L-TA R318M mutant is obtained by utilizing a molecular cloning technology, and the L-TA R318M mutant zymoprotein is obtained by constructing a GST fusion expression vector of the mutant gene and introducing the fusion expression vector into genetically engineered bacteria E.coli BL21 for induced expression. The L-TA R318M mutant is used as a catalyst, 3-/4-hydroxyl substituted benzaldehyde is used as a substrate, glycine is used as an auxiliary substrate, and 5-pyridoxal phosphate is used as a coenzyme, and an enzyme catalytic reaction is carried out in a proper condition and medium, so that the result shows that the diastereoselectivity of the mutant for synthesizing droxidopa (L-threo-DOPS) reaches 77.1 percent, which is more than 2.5 times of that of a wild enzyme, and the mutant has great application value in the industrial production of droxidopa.

The invention provides an L-threonine aldolase mutant which is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 3, and the mutant is obtained by changing Arg to Met at the 318 th amino acid of the L-threonine aldolase with the amino acid sequence of SEQ ID NO. 1.

The genes encoding the L-threonine aldolase mutants also belong to the scope of the present invention.

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

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

The vector provided by the invention can be a cloning vector, and comprises an L-TA R318M mutant gene and other elements required by plasmid replication; it may also be an expression vector comprising the L-TA R318M mutant gene and other elements enabling successful expression of the protein. In some embodiments of the invention, the expression vector is a pGEX-6p-2 vector into which the L-TA R318M mutant gene has been inserted, and the pGEX-6p-2 vector is a known commercially available vector.

The recombinant bacteria provided by the invention can be recombinant bacteria containing a cloning vector, such as E.coli DH5 alpha, and L-TA R318M mutant genes are replicated by culturing the recombinant bacteria; the recombinant bacterium may be a recombinant bacterium containing an expression vector, and the recombinant bacterium may be cultured under appropriate conditions to express a protein, for example: adding IPTG into the recombinant bacteria culture solution to induce the expression of L-TA R318M mutant protein at 16 deg.c.

The invention also provides a preparation method of the L-threonine aldolase mutant, which comprises the following steps: synthesizing the coding gene of the L-threonine aldolase mutant, constructing an expression vector, transforming protein expression host bacteria, inducing protein expression and purifying.

In a preferred embodiment of the production 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 pGEX-6p-2 vector and the protein expression host bacterium is E.coli BL21(DE 3).

The invention also provides a catalyst, the effective component of which comprises the L-threonine aldolase mutant. The catalyst can be used alone or with other suitable catalysts to improve the diastereoselectivity of the enzyme or to carry out two catalytic reactions in succession in the same reaction system.

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

In some preferred embodiments of the present invention, droxidopa is synthesized by using the L-threonine aldolase mutant or the catalyst and performing a catalytic reaction using 3, 4-dihydroxybenzaldehyde as a substrate, glycine as a co-substrate, and pyridoxal-5-phosphate as a coenzyme.

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

The L-TA R318M mutant can catalyze the aldol condensation reversible reaction of 3,4-dihydroxy benzaldehyde and glycine under the reaction conditions of 15-37 ℃ and pH 6.0-11.0.

Drawings

FIG. 1 is a schematic diagram of the catalytic synthesis of droxidopa by L-threonine aldolase (L-TA).

FIG. 2 shows the result of agarose gel electrophoresis of L-TA R318M mutant gene constructed by PCR; wherein M is a DNA Marker; R318M is a PCR product (L-TA R318M mutant gene).

FIG. 3 shows the double restriction enzyme digestion identification result of the recombinant plasmid pGEX-6p-2/L-TA R318M; wherein M is a DNA Marker; R318M is an enzyme cutting product.

FIG. 4 is an SDS-PAGE electrophoresis of L-threonine aldolase mutant R318M; wherein M is a protein molecular weight standard (Marker); R318M is mutant protein with molecular weight of about 38.7 KD.

FIG. 5 HPLC profile of the catalytic product of L-threonine aldolase mutant R318M; wherein the uppermost spectrum (labeled as R318M) shows the catalytic product of the L-TA R318M mutant, and the catalytic reaction is carried out at 25 ℃ and pH7.4 by using 3, 4-dihydroxybenzaldehyde as a substrate, glycine as an auxiliary substrate and pyridoxal 5-phosphate as a coenzyme; the intermediate spectrum (labeled L-threo-DOPS) is the L-threo-DOPS standard; the lowest spectra (labeled L-DOPS) are the L-threo-DOPS and L-erythro-DOPS racemate standards. The retention time of L-erythro-DOPS was 5.5min, and the retention time of L-threo-DOPS was 6.2 min.

Detailed Description

The invention is further described below in connection with specific examples, which are intended to be illustrative and illustrative only and not to limit the scope of the invention in any way.

Main reagents and consumables:

PrimeSTAR Max Premix (2 ×) (cat # R045A), BamH I restriction enzyme (cat # 1010S), Xho I restriction enzyme (cat # 1094S), T4 DNA Ligase (cat # D2011A) and 10 XT 4 Ligase buffer, all purchased from Taobao Biotech, Inc. (Dalian);

pGEX-6P-2 plasmid is known Escherichia coli expression vector, which is named as pGEX6P2 and pGEX6P2, the size of the vector is 4985bp, a Tac promoter, a vector label N-GST and vector resistance Ampicillin (Ampicillin), and is purchased from Shanghai Biotechnology limited company, and the laboratory also stores the plasmid;

trans5 α competent cells (cat # CD201-01) and E.coli BL21(DE3) competent cells (cat # CD601), both purchased from holo-gold biotechnology, Inc.;

glutamathione Sepharose 4B, available from GE Healthcare under cat No. 10223836;

PreScission Protease, available from GenScript, Inc., cat # Z02799-100;

bradford protein concentration assay kit, purchased from Beyotime corporation, cat # 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 racemate) standard (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, formula: c ₄ H ₈ O ₂ ) Are all purchased from carbofuran technologies, inc;

glycine (CAS: 56-40-6, molecular formula: C) ₂ H ₅ NO ₂ Cat # a502065), 3, 4-dihydroxybenzaldehyde (CAS: 139-85-5, linear formula: (HO) ₂ C ₆ H ₃ CHO, cat # A601406), pyridoxal 5-phosphate(English name PLP, CAS: 41468-25-1, molecular formula C) ₈ H ₁₂ NO ₇ P, cat # a610455), all purchased from bio-engineering (shanghai) incorporated.

LB Medium

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

The preparation method comprises the following steps: at 950ml ddH ₂ Dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride in O, adjusting pH to 7.4 with NaOH, and adding ddH ₂ And O is metered to 1L. If a solid medium is prepared, 15g of agar per liter are added. Sterilizing with high pressure steam at 121 deg.C for 20 min.

PBS buffer

preparation method of pH7.4, 10mM PBS: weighing 8g NaCl, 0.2g KCl and 1.44g Na ₂ HPO ₄ And 0.24g KH ₂ PO ₄ Dissolving in 800mL of distilled water, adjusting the pH value of the solution to 7.4 by using HCl, and finally adding distilled water to a constant volume of 1L. Sterilizing with steam at 121 deg.C for at least 20min, and storing in refrigerator at room temperature or 4 deg.C.

Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory grade. Unless otherwise specified, the methods used in the following examples are all conventional in the art, and the experimental conditions used are all conventional in the art, and reference may be made to the relevant experimental manuals or manufacturer's instructions.

Example 1 preparation of L-threonine Aldolase R318M mutant

Design of I, L-threonine aldolase mutant

The wild L-threonine aldolase gene (L-TA gene, SEQ ID NO:2) is isolated from feces samples of healthy black bears in Sichuan black bear protection and incubation bases in the laboratory, the total length of an open reading frame of the L-TA gene is 1032bp, and the encoded L-threonine aldolase consists of 343 amino acids. The isolation and cloning of this gene is described in the patent application text having application number 2019109657806, publication number CN110592058A, entitled "threonine aldolase, its encoding gene and use in droxidopa biosynthesis", which is incorporated herein by reference in its entirety.

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

MYSFKNDYSEGAHPRILETLLRTNLEQCEGYGKDTYCEEAENLIKNKLNNESIEVHFISGGTQTNLIAISAFLRPHEGVISADTGHIFVNEAGSIEATGHKVISVDVVDGKLRRDDILSVLSKFTNEHVVKPKLVYISNSTEIGTIYKKSELEELSKVCRENNLLLFMDGARLGSALSCKENDLTLEDISKLTDAFYIGGTKNGALLGEALVICNKDLQEDFRYHLKQKGAMLAKGRLLGIQFIELFKDDLFFEIGKHENDMADILRDGISRLGYEFLVDSPSNQIFPVFNNDIIRELEKNYGFNIWEKVNEEKTAIRLVTSFATKEEPCLEFIKFLSGLTNK(SEQ ID NO:1)

the nucleotide sequence (1032bp) of the coding gene of the wild-type L-threonine aldolase is as follows:

ATGTATAGTTTTAAAAATGATTATAGTGAAGGGGCACATCCTAGAATTCTTGAAACGTTGCTGAGAACAAATTTAGAACAATGTGAAGGTTACGGAAAAGATACATACTGTGAGGAAGCTGAAAACTTAATAAAAAATAAACTAAATAATGAGTCTATTGAAGTCCATTTCATATCTGGAGGTACACAAACTAACTTAATAGCAATATCTGCATTTTTAAGGCCTCATGAGGGTGTTATATCAGCAGATACAGGGCATATATTTGTAAATGAAGCAGGTTCAATAGAAGCAACAGGACATAAGGTGATATCTGTTGATGTTGTGGATGGTAAACTAAGAAGAGACGATATACTATCAGTATTGAGTAAGTTTACTAATGAGCATGTTGTAAAACCAAAGCTTGTTTATATATCTAACTCTACTGAAATTGGAACTATATATAAAAAATCTGAATTAGAAGAGTTAAGCAAAGTTTGTAGAGAAAATAATTTATTACTATTTATGGATGGAGCAAGATTAGGATCTGCACTTTCTTGCAAAGAAAATGATTTGACATTAGAAGATATAAGTAAATTAACTGATGCTTTTTATATCGGGGGAACTAAGAATGGAGCTCTTTTAGGAGAAGCACTTGTTATATGTAATAAAGATTTACAGGAAGATTTTAGATATCACTTAAAACAAAAAGGAGCGATGCTTGCTAAGGGAAGGTTGCTTGGAATACAGTTTATAGAATTATTTAAAGATGATTTATTTTTTGAAATAGGAAAACATGAAAATGATATGGCTGATATATTAAGGGATGGAATAAGTAGGCTTGGATATGAATTTTTAGTAGACTCTCCATCTAATCAAATATTCCCAGTATTTAACAATGATATTATAAGAGAATTAGAGAAAAACTATGGATTTAATATATGGGAAAAAGTAAATGAAGAGAAAACTGCAATAAGATTAGTAACATCTTTTGCAACAAAAGAAGAACCTTGTCTAGAGTTTATAAAGTTTTTAAGTGGATTAACTAATAAATAA(SEQ ID NO:2)

we determined that the site affecting the enzymatic properties is the 318 th amino acid of the wild-type L-threonine aldolase, which is arginine (R), and the corresponding nucleotide sequence is codon 952-954 by comparing the homology of the wild-type L-threonine aldolase with the homologous enzyme protein in a multi-layer system from primary structure to secondary structure. The codon 952-954 of the wild-type L-TA gene sequence is changed from AGA to ATG, so that the 318 th site of the amino acid sequence is replaced by the original arginine (R) to methionine (M), and the enzyme mutant is named as L-TA R318M mutant, the amino acid sequence of the enzyme mutant is shown as SEQ ID NO. 3, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.

II, obtaining L-TA R318M mutant gene

The L-TA R318M mutant gene can be obtained by a whole gene synthesis method or a molecular cloning method. The mutant gene was obtained by PCR.

1. Primer design

The obtained L-TA R318M mutant gene sequence (SEQ ID NO:4) was primed by adding BamH I (GGATCC) at the 5 'end and Xho I (CTCGAG) at the 3' end. The nucleotide sequences of the primers are as follows:

the upstream primer is as follows: L-TA-R318M-BamHI-F

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

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

5′-CCGCTCGAGTTATTTATTAGTTAATCCACTTAAAAACTTTATAAACTCTAGACAAGGTTCTTCTTTTGTTGCAAAAGATGTTACTAACATTATTG-3′(SEQ ID NO:6)。

The above primers were synthesized by the firm of Venezuelan Biotechnology engineering (Shanghai) Ltd.

2. Cloning of L-TA R318M mutant Gene

And (2) amplifying the L-TA R318M mutant gene by using a plasmid containing a wild type L-TA gene as a template and adopting the upstream primer and the downstream primer obtained in the step 1 according to the following PCR system and program. The method for preparing the plasmid containing the wild-type L-TA gene is described in the patent application No. 2019109657806, publication No. CN110592058A, entitled "threonine aldolase, its encoding gene and use in droxidopa biosynthesis", which is described as plasmid pGEX-6p-2/L-TA Sz-1-2 in example 3, the entire contents of which are incorporated herein by reference.

And (3) PCR system: PrimeSTAR Max Premix (2X) 25. mu.L, plasmid template 0.5. mu.L, upstream primer L-TA-R318M-BamHI-F (10. mu.M) 2. mu.L, downstream primer L-TA-R318M-XhoI-R (10. mu.M) 2. mu.L, 0.1% (g/ml) BSA 3. mu.L, complement ddH ₂ O to PCR system was 50. mu.L.

The PCR procedure was as follows:

a.94 ℃ for 5 min;

b.98 ℃ denaturation 10sec, 48 ℃ annealing 10sec, 72 ℃ extension 10 sec; 40 cycles;

c.72 ℃ extension for 10 min.

The PCR amplification product was detected by 1.2% agarose gel electrophoresis, and as a result, a band of about 1000bp in size was obtained as shown in FIG. 2. The band of interest was excised under an ultraviolet lamp, and the L-TA R318M mutant gene fragment was recovered using the Omega Gel Extraction Kit D2500 according to the Kit instructions.

3. Expression vector construction

(1) Cleavage and ligation

The L-TA R318M mutant gene and the pGEX-6p-2 vector are subjected to double enzyme digestion by using BamH I and Xho I restriction enzymes respectively. Enzyme cleavage System (Gene): 35 μ L of L-TA R318M mutant gene, 3 μ L of BamH I enzyme, 3 μ L of Xho I enzyme, 6 μ L of 10 XK buffer, and sterile double distilled water was supplemented until the system was 60 μ L. Enzyme digestion system (vector): 2 mu L of pGEX-6p-2 vector, 0.5 mu L of BamH I enzyme, 0.5 mu L of Xho I enzyme, 1 mu L of 10 xK buffer, and sterile double distilled water is supplemented until the system is 10 mu L. Enzyme cutting conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h.

The cleaved L-TA R318M mutant gene was then ligated with pGEX-6p-2 linear vector using T4 DNA ligase:

the ligation was carried out overnight at 16 ℃ to obtain a ligation product pGEX-6p-2/L-TA R318M.

(2) Transformation of

Trans5 alpha competent cells (gold, CD201-01) were placed on ice, 10. mu.L of pGEX-6p-2/L-TA R318M was added after the cells had thawed, and placed on ice for 30 min. The mixture was heat-shocked at 42 ℃ for 90 seconds and then kept on ice for 2 min. Adding 600 μ L sterile LB liquid medium, shaking at 37 deg.C and 150rpm for 45 min. Sucking 200 μ L of cultured bacterial liquid, and spreading on Amp ⁺ Resistant (100. mu.g/mL) LB plate medium and cultured overnight at 37 ℃ in an inverted state.

(3) Positive clone screening

Picking single colony on LB plate, inoculating to Amp ⁺ Resistant (100 mu g/mL) LB liquid medium, 37 ℃, 220rpm shake culture until OD600 ≈ 1.0, 8000rpm centrifugation 5min to collect thallus for plasmid extraction. The Plasmid was extracted using OMEGA Plasmid Mini Kit I (cat # D6943) according to the instructions and identified by double digestion.

Enzyme digestion system:

enzyme cutting conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h. The enzyme digestion product is detected by using 1.2 percent agarose gel electrophoresis, the recombinant plasmid with correct double enzyme digestion identification is selected and sent to TAKARA company for sequencing, and the recombinant plasmid with correct sequencing result is used as an expression vector of the L-TA R318M mutant.

4. GST fusion heterologous expression of enzyme proteins

(1) Coli BL21(DE3) cells

Coli BL21(DE3) competent cells were removed from-80 ℃ and placed on ice. After the cells were thawed, 10. mu.L of the expression vector pGEX-6p-2/L-TA R318M with the correct sequencing result was added and left on ice for 30 min. Heat shock was carried out at 42 ℃ for 90s, followed by standing on ice for 2 min. Adding 600 μ L sterile LB liquid medium, shaking at 37 deg.C and 150rpm for 45 min. Sucking 200 μ L of cultured bacterial liquid, and spreading on Amp ⁺ Resistant (100. mu.g/mL) LB plate medium and cultured overnight at 37 ℃ in an inverted state. And (3) picking a single colony on the LB plate, carrying out colony PCR identification according to the PCR system and the program in the step 2, and taking the colony with the correct identification result as a protein expression strain.

(2) Protein expression and purification

a. The protein expression strain was inoculated into a sterile LB liquid medium with a final ampicillin concentration of 50. mu.g/mL, cultured at 37 ℃ and 180 rpm.

b. When OD 600. apprxeq.0.8, IPTG was added to a final concentration of 0.2mM, and induction was carried out overnight (12h) at 16 ℃ and 180 rpm. The cells were collected by centrifugation at 8000rpm for 5 min.

c. The cells were resuspended in 1L of culture system in 30mL of lysis buffer (pH 7.4, 10mM PBS), and disrupted by sonication at 4 ℃ until clear. The crushed bacteria liquid is evenly distributed into a50 mL sterile centrifuge tube precooled at 4 ℃, centrifuged at 12000rpm for 20min at 4 ℃, and after centrifugation is finished, the supernatant is transferred into the 50mL sterile centrifuge tube precooled at 4 ℃ by a precision pipette gun.

d. Glutathione Sepharose 4B packing (GE Healthcare) was packed into a chromatography column (GE Healthcare) using 5mL of packing per liter of culture system. The absolute ethanol was removed by washing 3 column volumes with 4 ℃ pre-cooled pH7.4, 10mM sterile PBS. The supernatant from step c was bound to glutaminone Sepharose 4B for 2h at 4 ℃. The suspension was gently inverted vertically.

e. After the binding is completed, the filler is precipitated by centrifugation at 500rpm for 5 min. The packing was washed 3-5 column volumes with 4 ℃ pre-chilled pH7.4, 10mM sterile PBS to remove contaminating proteins.

f. The mixture was digested overnight at 4 ℃ with addition of a 4 ℃ precooled digestion buffer (pH 7.4, 10mM PBS) and PreScission Protease (GenScript, Z02799-100).

g. After the enzyme digestion is finished, discharging the supernatant from the chromatographic column to obtain L-TA R318M enzyme solution.

h. The molecular weight of the L-TA R318M enzyme solution is identified by SDS-PAGE detection. The concentration of L-TA R318M enzyme solution was determined using the Bradford protein concentration assay kit (Beyotime, P0006) according to the kit instructions.

The results are shown in FIG. 4, and the SDS-PAGE result shows that the L-TA R318M mutant is successfully expressed in a soluble way, the molecular weight of the protein is about 38.7KD, and the purity of the enzyme protein is high and is in a single band. The concentration of the 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 the de value

1. Synthesis of droxidopa

The substrate solution was prepared using PBS (pH 7.4, 10 mM) containing 1M glycine, 60mM 3, 4-dihydroxybenzaldehyde and 50. mu.M pyridoxal 5-phosphate. To each ml of the substrate solution, 20U of the L-TA R318M enzyme prepared in example 1 was added, mixed well, reacted at 25 ℃ for 2 hours, and then ultrafiltered (10kDa, 4000 Xg, 30 minutes) to obtain a sample.

2. Detection and de value calculation of droxidopa

And (3) diluting the sample obtained after ultrafiltration in the step (1) by 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 measured by HPLC. HPLC instrument model: agilent 1260. Setting parameters: a chromatographic column: COSMOSIL 5C18-MS 4.6X 150 mm; wavelength of 280nm and column temperature of 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; the sample volume was 10. mu.L, and the flow rate was 1 mL/min. L-threo-DOPS standards (0.1mg/mL, pH7.4, 10mM PBS), and racemic body standards of L-threo-DOPS and L-erythro-DOPS (0.01mg/mL, pH7.4, 10mM PBS), respectively, were tested according to the same HPLC parameters.

Determining the amount of L-threo-DOPS and L-erythro-DOPS in the sample based on the peak areas. The de value (percent diastereomeric excess) is 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) ]. times.100%.

The HPLC results are shown in FIG. 5, in which the uppermost spectrum (labeled R318M) is the catalytic product of the L-TA R318M mutant; the intermediate spectrum (labeled L-threo-DOPS) is the L-threo-DOPS standard; the lowest spectra (labeled L-DOPS) are the standards for L-threo-DOPS and L-erythro-DOPS racemates. The retention time of L-erythro-DOPS was 5.5min, and the retention time of L-threo-DOPS was 6.2 min. The L-TA R318M mutant was calculated to have 77.1% diastereoselectivity (de value) in catalyzing the synthesis of droxidopa (L-threo-DOPS) from 3, 4-dihydroxybenzaldehyde and glycine.

Sequence listing

<110> university of Chongqing

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

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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 Arg 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> 2

<211> 1032

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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 taaagttttt aagtggatta 1020

actaataaat aa 1032

<210> 3

<211> 343

<212> PRT

<213> Artificial Sequence (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 (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 taaagttttt aagtggatta 1020

actaataaat aa 1032

<210> 5

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgcggatcca tgtatagttt taaaaatgat tata 34

<210> 6

<211> 95

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccgctcgagt tatttattag ttaatccact taaaaacttt ataaactcta gacaaggttc 60

ttcttttgtt gcaaaagatg ttactaacat tattg 95

Claims

1. An L-threonine aldolase mutant characterized in that the amino acid sequence thereof is represented by SEQ ID NO. 3, and the amino acid at position 318 of the L-threonine aldolase having the amino acid sequence of SEQ ID NO. 1 is changed from Arg to Met.

2. A gene encoding the L-threonine aldolase mutant as described in claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is represented by SEQ ID NO. 4.

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

5. The method for producing an L-threonine aldolase mutant according to claim 1, comprising the steps of: synthesizing the coding gene of the L-threonine aldolase mutant, constructing an expression vector, transforming protein expression host bacteria, inducing protein expression and purifying.

6. The method of claim 5, wherein: the nucleotide sequence of the coding gene of the L-threonine aldolase mutant is shown as SEQ ID NO. 4.

7. A catalyst comprising the L-threonine aldolase mutant according to claim 1 as an active ingredient.

8. Use of the L-threonine aldolase mutant as defined in claim 1 or the catalyst as defined in claim 7 in an aldol condensation reaction.

9. The use according to claim 8, wherein droxidopa is synthesized by a catalytic reaction 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 co-substrate, and pyridoxal-5-phosphate as a co-enzyme.

10. Use according to claim 9, wherein the catalytic reaction is carried out at a temperature of 15-37 ℃ and a pH of 6.0-11.0.