CN114703169A

CN114703169A - L-threonine aldolase mutant R318L/H128N and application thereof

Info

Publication number: CN114703169A
Application number: CN202210466832.7A
Authority: CN
Inventors: 戚娜; 王伯初; 马劲松
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2022-07-05
Anticipated expiration: 2042-04-29
Also published as: CN114703169B

Abstract

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

Description

L-threonine aldolase mutant R318L/H128N and application thereof

Technical Field

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

Background

Droxidopa (L-threo-DOPS, i.e. L-threo- (3, 4-dihydroxy) phenylserine) is the only drug used to treat parkinson-related Neurogenic Orthostatic Hypotension (NOH) since the approval of midodrin in 1996, for the improvement of gait rigidity and orthostatic dizziness caused by parkinson's 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 the troxidopa comprises chemical synthesis and enzyme catalysis, and the industrial production of the troxidopa depends on the chemical synthesis. 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 virulent hydrogen sulfide are used in the reaction process, and the optical resolution causes half of raw material waste, high production cost and potential environmental pollution, and 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.

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. 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 by using benzaldehyde and glycine substituted by hydroxyl at 3-/4-position as substrates is 30%. The low diastereoselectivity greatly hinders the use of this enzyme in the synthesis of troxidopa. Therefore, it is very necessary to develop threonine aldolase with high selectivity for the synthesis of droxidopa.

Disclosure of Invention

Has important influence on the stereoselectivity and the catalytic activity of threonine aldolase

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 in a multi-layer system, under the premise that the site influencing the optical property of the wild-type L-threonine aldolase is determined to be arginine at the 318 th site in the former period and arginine (Arg) at the site is changed into leucine (Leu), amino acid His128 with subunit interaction is discovered through semi-rational design and rational design, and the amino acid His at the 128 th site is changed into Asn, so as to obtain an enzyme mutant which is named as L-TA R318L/H128N mutant. The encoding gene (SEQ ID NO:4) of the L-TA R318L/H128N mutant is obtained by utilizing a molecular cloning technology, and the L-TA R318L/H128N mutant enzyme protein is obtained by constructing a GST fusion expression vector of the mutant gene and introducing the GST fusion expression vector into genetically engineered bacterium E.coli BL21 for induction expression. The L-TA R318L/H128N 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) is as high as 92.89 percent, which is 3.09 times of that of a wild type, the yield reaches 1.26mg/mL, 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 is shown as SEQ ID NO. 3, and the mutant is obtained by changing Arg to Leu at the 318 th amino acid position and changing His to Asn at the 128 th amino acid position 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 R318L/H128N mutant gene and other elements required by plasmid replication; it may also be an expression vector comprising the L-TA R318L/H128N 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 R318L/H128N mutant gene is 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 R318L/H128N mutant genes are replicated by culturing the recombinant bacteria; or a recombinant bacterium containing an expression vector, such as e.coli BL21, cultured under appropriate conditions and expressed as a protein, such as: adding a proper amount of IPTG into the recombinant bacterium culture solution, and inducing the expression of the L-TA R318L/H128N mutant protein at 16 ℃.

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 together with other suitable catalysts to improve the diastereoselectivity of the enzyme or to carry out two catalytic reactions one after the other 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 invention, the catalytic reaction is carried out at a temperature of 15 to 37 ℃ and a pH of 6.0 to 11.0.

The L-TA R318L/H128N 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 is an 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, and the molecular weight of the mutant protein is about 38.7 KD.

FIG. 3 HPLC profile of the catalytic product of L-threonine aldolase mutant R318L/H128N; wherein the uppermost spectrum (labeled as R318L/H128N) shows the catalytic product of the L-TAR318L/H128N mutant, and the catalytic reaction is carried out at 25 ℃ and pH 7.4 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 to be construed as merely illustrative and explanatory and not limiting the scope of the invention in any way.

Main reagents and consumables:

prime STAR 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 baoz biotechnology limited (da lian);

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 has storage;

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

glutaminone Sepharose 4B, purchased from GE Healthcare, cat # 10223836;

PreScission Protease, available from Gen Script Inc., cat No. 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)₉H₁₁NO₅) LDOPS (L-threo-DOPS and L-erythro-DOPS racemate) standards (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: c₄H₈O₂) Are all purchased from carbofuran technologies, inc;

glycine (CAS: 56-40-6, molecular formula: C)₂H₅NO₂No. a502065), 3, 4-dihydroxybenzaldehyde (CAS: 139-85-5, linear formula: (HO)₂C₆H₃CHO, cat # a601406), pyridoxal 5-phosphate (english name PLP, CAS: 41468-25-1, formula C₈H₁₂NO₇P, 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 ddH₂O10 g tryptone, 5g yeast extract, 10g sodium chloride, then adjusted to pH 7.4 with NaOH, and ddH₂And 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 pH 7.4, 10mM PBS: weighing 8g NaCl, 0.2g KCl and 1.44g Na₂HPO₄And 0.24g KH₂PO₄Dissolving the mixture 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 R318L/H128N 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 text of the patent application with application No. 2019109657806, publication No. 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(SEQIDNO:1)

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

ATGTATAGTTTTAAAAATGATTATAGTGAAGGGGCACATCCTAGAATTCTTGAAACGTTGCTGAGAACAAATTTAGAACAATGTGAAGGTTACGGAAAAGATACATACTGTGAGGAAGCTGAAAACTTAATAAAAAATAAACTAAATAATGAGTCTATTGAAGTCCATTTCATATCTGGAGGTACACAAACTAACTTAATAGCAATATCTGCATTTTTAAGGCCTCATGAGGGTGTTATATCAGCAGATACAGGGCATATATTTGTAAATGAAGCAGGTTCAATAGAAGCAACAGGACATAAGGTGATATCTGTTGATGTTGTGGATGGTAAACTAAGAAGAGACGATATACTATCAGTATTGAGTAAGTTTACTAATGAGCATGTTGTAAAACCAAAGCTTGTTTATATATCTAACTCTACTGAAATTGGAACTATATATAAAAAATCTGAATTAGAAGAGTTAAGCAAAGTTTGTAGAGAAAATAATTTATTACTATTTATGGATGGAGCAAGATTAGGATCTGCACTTTCTTGCAAAGAAAATGATTTGACATTAGAAGATATAAGTAAATTAACTGATGCTTTTTATATCGGGGGAACTAAGAATGGAGCTCTTTTAGGAGAAGCACTTGTTATATGTAATAAAGATTTACAGGAAGATTTTAGATATCACTTAAAACAAAAAGGAGCGATGCTTGCTAAGGGAAGGTTGCTTGGAATACAGTTTATAGAATTATTTAAAGATGATTTATTTTTTGAAATAGGAAAACATGAAAATGATATGGCTGATATATTAAGGGATGGAATAAGTAGGCTTGGATATGAATTTTTAGTAGACTCTCCATCTAATCAAATATTCCCAGTATTTAACAATGATATTATAAGAGAATTAGAGAAAAACTATGGATTTAATATATGGGAAAAAGTAAATGAAGAGAAAACTGCAATAAGATTAGTAACATCTTTTGCAACAAAAGAAGAACCTTGTCTAGAGTTTATAAAGTTTTTAAGTGGATTAACTAATAAATAA(SEQIDNO:2)

by comparing the homology of the wild-type L-threonine aldolase and the homologous enzyme protein from the primary structure to the high-level structure in a multi-layer system from multi-angle, the sites influencing the enzymatic properties are determined to be the 318 th amino acid and the 128 th amino acid of the wild-type L-threonine aldolase, and the corresponding nucleotide sequences are the 952-. The codon 952-954 of the wild-type L-TA gene sequence was changed from AGA to CTG and the codon 382-384 was changed from GCT to AAT, so that the 318 th position of the amino acid sequence was changed from the original arginine (R) to leucine (L) and the original histidine (H) to asparagine (N), to obtain an enzyme mutant, named L-TA R318L/H128N mutant, whose amino acid sequence is shown in SEQ ID NO:3 and whose nucleotide sequence of the encoding gene is shown in SEQ ID NO: 4.

II, obtaining L-TA R318L/H128N mutant gene

The L-TA R318L/H128N 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 R318L/H128N 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 L-TA-R318L-BamH I-F:

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

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

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

the upstream primer L-TA-R318L/H128N-F:

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

the downstream primer L-TA-R318L/H128N-R:

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

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

2. L-TA R318L/H128N mutant gene clone

The invention is named as follows under the application number of 202010323801.7: the plasmid of the L-TA mutant R318L gene reported in the section "L-threonine aldolase mutant R3318L and its application" was used as a template, and the upstream primer and the downstream primer obtained in step 1 were used to amplify the L-TA R318L/H128N mutant gene according to the following PCR system and procedure.

And (3) PCR system: prime STAR Max Premix (2X) 25. mu.L, plasmid template 0.5. mu.L, upstream primer L-TA R318L/H128N-BamH I-F (10. mu.M) 2. mu.L, downstream primer L-TA-R318L/H128N-Xho I-R (10. mu.M) 2. mu.L, complement ddH₂O to a total volume of 50. mu.L.

The PCR procedure was as follows:

a.94 ℃ for 5 min;

b.98 ℃ denaturation 10sec, 47 ℃ 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 to obtain a band of about 1000bp in size. The band of interest was excised under an ultraviolet lamp, and the L-TA R318L/H128N 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 R318L/H128N 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 R318L/H128N mutant gene, 3 μ L of BamH I enzyme, 3 μ L of Xho I enzyme, and 10 XK buffer6 μ L, and sterile double distilled water is supplemented until the system is 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. The enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h.

The cleaved L-TA R318L/H128N mutant gene was then ligated with pGEX-6p-2 linear vector using T4 DNA ligase: 6 mu L of the L-TA R318L/H128N mutant gene after enzyme digestion, 2 mu L of pGEX-6p-2 linear vector, 1 mu L of T4 DNA Ligase and 1 mu L of 10 XT 4 Ligase buffer. Ligation was carried out overnight at 16 ℃ to give the ligation product pGEX-6p-2/L-TA R318L/H128N.

(2) Transformation of

Trans5 alpha competent cells (gold, CD201-01) were placed on ice, 10. mu.L of pGEX-6p-2/L-TA R318L/H128N were 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. 200. mu.L of the cultured bacterial suspension was aspirated, spread on Amp + resistant (100. mu.g/mL) LB plate medium, and cultured overnight at 37 ℃ in an inverted manner.

(3) Positive clone screening

A single colony on an LB plate is picked up and inoculated in an Amp + resistant (100 mu g/mL) LB liquid medium, shake-cultured at 37 ℃ and 220rpm until OD 600 is approximately equal to 1.0, and centrifuged at 8000rpm for 5min to collect thalli 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 products of the restriction enzyme digestion are detected by using 1.2 percent agarose gel electrophoresis, the recombinant plasmid with correct restriction 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 R318L/H128N mutant.

4. GST fusion heterologous expression of enzyme proteins

(1) Coli BL21(DE3) cells transformed with the plasmid

Coli BL21(DE3) competent cells were removed from-80 ℃ and placed on ice. After the cells are thawed, 10. mu.L of expression vector pGEX-6p-2/L-TA R318L/H128N with correct sequencing result is added, and the mixture is 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. 200. mu.L of the cultured bacterial suspension was aspirated, spread on Amp + resistant (100. mu.g/mL) LB plate medium, and cultured overnight at 37 ℃ in an inverted manner. 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 to a final concentration of 100. mu.g/mL ampicillin, and cultured at 37 ℃ and 170 rpm.

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

c. The cells were resuspended in a 1L culture system in 30mL of lysis buffer (pH 7.4, 10mM PBS), and the cell walls were disrupted by an ultra-high pressure nano homogenizer at 4 ℃. 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-chilled pH 7.4, 10mM sterile PBS. The supernatant from step c was combined with glutaminone Sepharose 4B and suspended vertically for 3h at 4 ℃.

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 pH 7.4, 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 the L-TAR318L/H128N enzyme solution.

h. The molecular weight of the L-TA R318L/H128N enzyme solution is identified by SDS-PAGE. The concentration of L-TA R318L/H128N enzyme solution was determined using the Bradford protein concentration assay kit (Beyotime, P0006) according to the kit instructions.

The results are shown in FIG. 2, and the SDS-PAGE result shows that the L-TA R318L/H128N 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 a single band. The concentration of the L-TA R318L/H128N enzyme solution was determined to be 2 mg/mL.

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

1. Synthesis of droxidopa

After adding 1M glycine, 60mM 3, 4-dihydroxybenzaldehyde and 50. mu.M PLP, 2.8mg of purified L-TA to PBS buffer (pH 7.0), and reacting at 37 ℃ for 4 hours, the sample was ultrafiltered with an ultrafiltration tube (10kDa) to remove insoluble matter. The ultrafiltered sample was lyophilized in a lyophilizer, and then an organic phase solution (90% 0.1% w/v sodium heptanesulfonate/0.1% w/v 1, 4-dioxane) was added to bring to the appropriate concentration.

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 number: agilent 1260. Setting parameters: a chromatographic column: COSMOSIL5C18-MS 4.6X 150 mm; the wavelength is 280nm, and the column temperature is 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, pH 7.4, 10mM PBS), and racemic standards of L-threo-DOPS and L-erythro-DOPS (0.01mg/mL, pH 7.4, 10mM PBS), respectively, were tested according to the same HPLC parameters.

Determining the amounts 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. 3, in which the uppermost spectrum (labeled R318L/H128N) is the catalytic product of the L-TAR318L/H128N mutant; the intermediate spectrum (labeled L-threo-DOPS) is the L-threo-DOPS standard; the lowest spectrum (labeled L-DOPS) is the standard 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-erythro-DOPS was 6.2 min. The L-TAR318L/H128N mutant was calculated to have a diastereoselectivity (de value) of 84.9% in catalyzing the synthesis of droxidopa (L-threo-DOPS) from 3, 4-dihydroxybenzaldehyde and glycine.

Claims

1. An L-threonine aldolase mutant characterized in that the amino acid sequence is shown as SEQ ID NO. 3, and the mutant is obtained by changing Arg to Leu at amino acid 318 and changing His to Asn at amino acid 128 of the L-threonine aldolase with the amino acid sequence of SEQ ID NO. 1.

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.