CN110592058A

CN110592058A - Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis

Info

Publication number: CN110592058A
Application number: CN201910965780.6A
Authority: CN
Inventors: 赵文艳; 杨碧玲; 潘银平; 倪德春; 王伯初; 戚娜; 祝连彩
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2019-05-30
Filing date: 2019-10-12
Publication date: 2019-12-20
Anticipated expiration: 2039-10-12
Also published as: CN110592058B

Abstract

The invention relates to an L-threonine aldolase, a coding gene and application thereof, belonging to the technical field of biology. The L-threonine aldolase is a protein shown in SEQ ID NO.1, or a protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in SEQ ID NO. 1. The L-threonine aldolase can perform enzyme catalytic reaction in a proper condition and medium by using 3-/4-hydroxyl-substituted benzaldehyde as a substrate, pyridoxal 5-phosphate as a coenzyme and glycine as an auxiliary substrate to biosynthesize droxidopa (L-threo-DOPS), and the diastereoselectivity of the method reaches 30%. The L-threonine aldolase with higher selectivity has important significance for droxidopa biocatalysis.

Description

Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis

Technical Field

The invention relates to the field of biotechnology, in particular to L-threonine aldolase, a coding gene thereof and application thereof in droxidopa biosynthesis.

Background

Droxidopa (L-threo-DOPS) is a novel anti-Parkinson disease drug. For improving gait stiffness 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 droxidopa mainly comprises chemical synthesis and enzymatic catalysis. The chemical synthesis method mainly obtains chiral droxidopa through addition reaction, esterification reaction and chemical resolution, although the chemical method is simple to operate, 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 conditions, small 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 (TA) 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 α carbon of the threonine substrate. However, the stability of threonine aldolase is poor, and the chiral selectivity, especially the chiral selectivity of beta-hydroxy is insufficient, so that the application of the threonine aldolase in chiral synthesis is limited. It is therefore highly necessary to develop threonine aldolases with high selectivity for the synthesis of droxidopa.

Disclosure of Invention

The invention utilizes metagenome sequencing technology to separate a new L-threonine aldolase gene from excrement samples of healthy black bears in Sichuan black bear protection and incubation bases, the gene is named as L-TA Sz-1-2 gene, the nucleotide sequence of the gene is shown as a sequence 2 in a sequence table, an open reading frame is arranged from the 1 st position to the 1032 th position of the 5' end, the length is 1032bp, and the coded amino acid of the L-TA Sz-1-2 protein is shown as a sequence 1 and consists of 343 amino acid sequences.

The L-threonine aldolase belongs to a biocatalyst, can use benzaldehyde substituted by 3-/4-hydroxyl as a substrate, pyridoxal 5-phosphate as a coenzyme and glycine as an auxiliary substrate to carry out enzyme catalytic reaction in a proper condition and medium, and is used for biosynthesizing droxidopa (L-threo-DOPS), and the diastereoselectivity of the method can reach 30%. Because droxidopa is mainly synthesized by chemical synthesis under the harsh conditions and the enzyme catalysis does not realize industrial production due to a low de value, the L-threonine aldolase has important significance for the biological catalysis of droxidopa.

In one aspect, the present invention provides an L-threonine aldolase represented by a1 or a 2:

a protein shown as SEQ ID NO. 1;

a2. the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

The invention also provides a coding gene of the L-threonine aldolase. Preferably, the coding gene is any one of the following b1-b 3:

b1. a DNA molecule represented by SEQ ID NO. 2;

b2. a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in b1 and which encodes said L-threonine aldolase;

b3. a DNA molecule having 90% or more identity to the DNA molecule defined in b1 or b2 and encoding said L-threonine aldolase.

Recombinant vectors, expression cassettes or recombinant cells containing said coding genes also belong to the scope of protection of the present invention. The vector can be a cloning vector and comprises an L-TA Sz-1-2 gene and other elements required for plasmid replication; it may also be an expression vector comprising the L-TA Sz-1-2 gene and other elements that enable successful expression of the protein. Recombinant cells refer to transgenic cell lines or recombinant bacteria, and may be recombinant cells comprising a cloning vector, e.g., e.coli DH5 α; or a recombinant cell comprising an expression vector, such as e.coli BL 21. The expression of L-TA Sz-1-2 can be induced by culturing the recombinant cells under appropriate conditions.

The application of the L-threonine aldolase in catalytic reaction also belongs to the protection scope of the invention.

The invention also provides a catalyst, which comprises the L-threonine aldolase. The catalyst may be used simultaneously with other suitable catalysts to increase the efficiency of the enzyme catalysis or to carry out two catalytic reactions one after the other in the same reaction system.

In another aspect, the present invention provides a method for effecting selective aldehyde condensation of a substrate by using the L-threonine aldolase or the catalyst to catalyze the reaction of the substrate.

Preferably, the catalytic reaction takes 3, 4-dihydroxybenzaldehyde as a substrate, takes pyridoxal-5-phosphate as a coenzyme and glycine as an auxiliary substrate to synthesize droxidopa.

Preferably, the amount of L-threonine aldolase is 100-2000mg/mL, the concentration of 3, 4-dihydroxybenzaldehyde is 1-100g/L, the concentration of glycine is 5-100g/L, and the concentration of pyridoxal-5-phosphate is 0.01-0.2 g/L.

Preferably, the temperature of the catalytic reaction is controlled to be 5-42 ℃, and the stirring speed is 70-300 rpm.

Drawings

FIG. 1 is a schematic diagram of the selective synthesis of droxidopa by L-threonine aldolase.

FIG. 2 is an agarose gel electrophoresis image of the isolated L-TA Sz-1-2 gene.

FIG. 3 is an SDS-PAGE electrophoresis of L-TA Sz-1-2 protein, wherein lane 1 is a Marker of 14kDa to 120kDa, and lane 2 is L-TA Sz-1-2 protein.

FIG. 4 shows the alignment of L-TA Sz-1-2 with the existing E.coli L-threonine aldolase.

FIG. 5 is an HPLC chromatogram of synthetic droxidopa with peak 1 being L-erythro-DOPS and peak 2 being L-threo-DOPS (droxidopa).

FIG. 6 shows the effect of pH on de-value of the L-TA Sz-1-2 enzymatic reaction.

FIG. 7 shows the effect of temperature on de value of the L-TA Sz-1-2 enzymatic reaction.

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.

Biological material：

Coli DH5 α competent cells, purchased from tiangen biochemical technology (beijing) ltd, cat #: CB 101;

coli BL21 competent cells, purchased from tiangen biochemical technology (beijing) ltd, cat #: CB 105.

Experimental reagent：

Fecal DNA genome extraction kit, purchased from Qiagen, germany, cat #: 51604, respectively;

prime STAR HS DNA Polymerase, purchased from TaKaRa, cat # stock: DR 010A;

agarose gel recovery kit, purchased from Omega, cat No.: d2500-01;

vector pGEX-6p-2, purchased from Youbao: cargo number VT 1259;

plasmid extraction Kit OMEGA Plasmid Mini Kit I, purchased from OMEGA, cat #: d6943;

restriction enzyme BamHI, purchased from gangbao biotechnology limited, cat #: 1010S;

restriction enzyme XhoI, purchased from gangbao biotechnology limited, cat #: 1094S;

t4DNA Ligase, purchased from british biotechnology limited, cat #: D2011A; 10 XT 4Ligase buffer, purchased from Dalianbao Biotechnology Ltd;

lysis buffer: prepared, 10mM PBS with pH 7.3 contains PMSF 0.1mM and Leupeptin 0.5 mg/mL;

glutaminone Sepharose 4B, purchased from GE Healthcare, cat No.: 10223836, respectively;

PreScission Protease, purchased from GenScript, Cat. No.: z02799-100;

BCA kit, purchased from Beyotime, cat # s: p0006;

L-threo-DOPS, available from Profenor technologies, Cathaka: d4235;

glycine, purchased from bio-engineering (shanghai) gmbh, cat #: a502065;

3, 4-dihydroxybenzaldehyde, purchased from bio-engineering (shanghai) gmbh, cat #: a601406;

PLP, purchased from bio-engineering (shanghai) gmbh, cat #: a610455;

EB, purchased from carbofuran technologies, cat #: 242101, respectively;

the black bear feces used for DNA extraction are frozen and stored in the laboratory.

The biochemical reagents not specifically described in the following examples are all conventional reagents in the art, can be prepared according to conventional methods in the art or are commercially available, and are all chemically pure and biologically pure.

Example 1 discovery of L-TA Sz-1-2 Gene

Using the sterilized medicine spoon, taking the feces sample of the healthy black bear in the Sichuan black bear protection and incubation base, preserving the feces sample with dry ice and transporting the feces sample back to the laboratory. Total DNA of the black bear feces is extracted by using a Qiagen feces DNA genome extraction kit, and the extracted total DNA is handed to Megji biological medicine science and technology Limited in Shanghai for metagenome sequencing. A new L-threonine aldolase gene is found by comparing the existing coding gene sequence (Genbank accession number: AOM47009.1) of Escherichia coli L-threonine aldolase (E.coli. L-TA) with metagenome sequencing data, and is named as L-TA Sz-1-2, and the nucleotide sequence of the L-threonine aldolase gene is shown as a sequence 2. The 1 st site to the 1032 th site of the 5' end of the sequence 2 are open reading frames, the open reading frame portion is 1032bp, and the amino acid of the encoded L-TA Sz-1-2 protein is shown as the sequence 1 and consists of 343 amino acids. The sequence alignment showed that the identity of this novel L-TA Sz-1-2 gene to the existing E.coli L-threonine aldolase gene sequence was 69.86% (FIG. 4).

Example 2 cloning of L-TA Sz-1-2 Gene

1) Designing a primer: designing a primer for the sequence of the L-TA Sz-1-2 gene obtained by sequencing, wherein the nucleotide sequence of the primer is as follows:

Sz-1-2-BamHI-F：5′-CGCGGATCCATGTATAGTTTTAAAAATGATTATA-3' (SEQ ID NO: 3 in the sequence listing),

Sz-1-2-XhoI-R：5′-CCGCTCGAGTTATTGTACTTCATCTAATAGCAT-3' (SEQ ID NO: 4 in the sequence listing).

2) And (3) PCR amplification: and (2) taking the total DNA of the black bear feces as a template, adopting the primer obtained in the step 1), and carrying out amplification according to the following PCR system and program.

And (3) PCR system: the template DNA was 0.5. mu.L, Sz-1-2-BamHI-F (10. mu.M) was 2. mu.L, Sz-1-2-XhoI-R (10. mu.M) was 2. mu.L, 0.1% BSA was 3. mu.L, Prime STAR HS DNA Polymerase was 25. mu.L, and ddH was supplemented₂O to PCR system was 50. mu.L.

The PCR procedure was as follows:

a.94 ℃ for 5 min;

denaturation at 98 ℃ for 10sec, annealing at 48 ℃ for 10sec, extension at 72 ℃ for 10sec, 40 cycles;

c.72 ℃ extension for 10 min.

3) Agarose gel electrophoresis: the PCR amplification product was detected by 1.2% agarose gel electrophoresis, and as a result, a band of about 1000bp in size was observed as shown in FIG. 2.

4) Cutting and recycling the rubber: the band of interest was cut under an ultraviolet lamp, and the gene fragment of interest was recovered using an Omega agarose gel recovery kit according to the procedures described in the kit instructions.

5) Double restriction enzyme digestion of L-TA Sz-1-2 Gene: BamHI and XhoI (both from Dalianbao Biotechnology Co., Ltd.) were used as restriction enzymes, and the L-TA Sz-1-2 gene fragment recovered by gel cutting in step 4) was digested by double digestion.

The double enzyme digestion system comprises: the L-TA Sz-1-2 gene was 35. mu.L, BamHI 3. mu.L, XhoI 3. mu.L, 10 XK buffer 6. mu.L, sterile double distilled water was supplemented to 60. mu.L. The enzyme was cleaved at 37 ℃ for 3 hours.

6) Recovering a double enzyme digestion product: and (3) recovering the double-enzyme digestion target gene fragment by using an Omega recovery kit according to the operation steps in the kit instruction.

Example 3 expression, extraction and purification of L-TA Sz-1-2 Gene

1. Constructing a connecting vector: the full sequence of the L-TA Sz-1-2 gene is connected to the vector pGEX-6 p-2.

The connection system comprises: the double restriction enzyme digestion L-TA Sz-1-2 gene is 6. mu.L, the T4DNA Ligase is 1. mu.L, the vector pGEX-6p-2 is 2. mu.L, the 10 XT 4Ligase buffer is 1. mu.L, the system is 10. mu.L, and the ligation is carried out at 16 ℃ for 12 h.

2. Coli DH5 α competent cells, procedure was as follows:

1) coli DH5 α were thawed on ice.

2) The ligation system obtained in step 1 was added to the thawed e.coli DH5 α competence on ice for 30 min.

3) Heat treatment at 42 deg.C for 90 s.

4) Standing on ice for 2 min.

5) Adding 600 μ L of non-resistant LB medium, shaking at 37 deg.C for 45min, and shaking at 150 rpm.

6) 200. mu.L of the bacterial solution was aspirated and applied to ampicillin (Amp)⁺) Resistant LB plate medium.

7) The culture was carried out overnight at 37 ℃.

3. And (3) identifying positive clones by the following steps:

1) selecting single colony, inoculating on LB/Amp⁺The liquid medium (4) was cultured at 37 ℃ and 180rpm for 12 hours, and centrifuged at 14000rpm for 4min to obtain cells.

2) The Plasmid was extracted using the OMEGA Plasmid Mini Kit I Plasmid extraction Kit according to the procedures described in the specification.

3) PCR verification was performed using the cloning primers Sz-1-2-BamHI-F and Sz-1-2-XhoI-R according to the PCR system and procedure of example 2 to confirm successful insertion of the gene fragment into the pGEX-6p-2 vector.

4) The recombinant plasmid with correct identification is sent to Shanghai bio-chemical company for sequencing, and the recombinant plasmid pGEX-6p-2/L-TA Sz-1-2 with correct sequencing result comparison is used as a gene expression vector.

Expression of L-TA Sz-1-2

1) Coli BL21 cells transformed with the plasmid

a. Coli bl21 competent cells were removed at-80 ℃ and placed on ice for 10 min.

b. Adding 2 μ L expression vector pGEX-6p-2/L-TA Sz-1-2, and standing on ice for 30 min.

c.42 ℃ for 90 sec.

d. Standing on ice for 2 min.

e. After recovery, 600. mu.L of LB medium was added and shaking cultured at 37 ℃ and 150rpm for 45 min.

f. Sucking 200 μ L of bacterial liquid, and coating on Amp⁺LB plate medium.

g.37 ℃ for overnight culture to obtain the recombinant cells.

2) Recombinant cell expression and purification

a. The recombinant cells were inoculated into sterile LB liquid medium with a final ampicillin concentration of 50. mu.g/mL, shake-cultured at 37 ℃ and 180 rpm.

b. When the OD600 of the bacterial liquid is about 0.8, isopropyl thiogalactoside (IPTG) with the final concentration of 0.5mM is added, and the induction is carried out for 12h at the temperature of 16 ℃. The cells were collected at 8000rpm for 5 min.

c. Resuspend the thallus according to the proportion of adding 30mL lysine buffer into 1L culture system, and break the thallus by ultrasound until the thallus is clear. Centrifuge at 12000rpm for 20 min. And taking the supernatant.

d. The supernatant was combined with glutaminone Sepharose 4B using 5mL of filler per liter of culture system and combined for 2h at 4 ℃. The suspension was gently inverted vertically.

e. After the completion of the bonding, the filler was precipitated at 5000rpm for 5 min. The packing was washed 3-5 column volumes with 4 ℃ pre-chilled PBS to remove contaminating proteins.

f. Add PreScission Protease digestion buffer.

g.4 ℃ overnight. After the enzyme digestion is finished, the supernatant is discharged from the chromatographic column.

h. The resulting samples were subjected to SDS-PAGE to determine their molecular weight and purity, which was about 38.7kD, and the concentration of purified protein was determined using the BCA kit according to the protocol described in the kit instructions.

As shown in FIG. 3, the obtained L-TA Sz-1-2 enzyme protein had a high purity and a single band. In FIG. 3, lane 1 shows the 14kDa-120kDa protein molecular marker, and lane 2 shows L-TA Sz-1-2.

Example 4 determination of optimum pH value of L-TA Sz-1-2

pH buffers configured for different pH gradients: pH 6.0-8.0: 50mM sodium phosphate buffer; pH 8.0-9.0: 50mM Tris-HCl; pH 9.0-11: 50mM glycine-NaOH, 0.5 pH values per gradient interval. The optimum pH for the maximum selectivity for the synthesis of droxidopa by L-threonine aldolase at pH 6.0-11.0 was determined.

De values (diasterogenic excesses, percent diastereomeric excess) were determined: [ (L-threo-DOPS-L-erythro-DOPS)/(L-threo-DOPS + L-erythro-DOPS) ]. 100%

The catalytic reaction was carried out using the L-threonine aldolase obtained in example 3 with 3, 4-dihydroxybenzaldehyde as a substrate, pyridoxal-5-phosphate as a coenzyme, and glycine as an auxiliary substrate, with the temperature controlled at 5-42 ℃ and the stirring speed at 70-300rpm, to synthesize droxidopa. In the catalytic reaction system, the dosage of the L-threonine aldolase is 100-2000mg/mL, the concentration of the 3, 4-dihydroxybenzaldehyde is 1-100g/L, the concentration of the glycine is 5-100g/L, and the concentration of the pyridoxal-5-phosphate is 0.01-0.2 g/L. Droxidopa and its diastereoisomers were detected by HPLC with HPLC parameters set to: wavelength 280nm, column temperature 30 ℃, methanol: 0.1% (W/V) sulfonic acid heptane buffer salt ═ 90: 10, flow rate 1 mL/min. The diastereoselectivity was calculated by peak area.

As shown in FIG. 6, the optimum pH of the L-TA Sz-1-2 of the present invention falls within a relatively wide range, and the L-TA Sz-1-2 of the present invention has a high diastereoselectivity at pH6.0 to 11, and has a diastereoselectivity (de value) in the range of 28% to 32%, indicating that the L-TA Sz-1-2 of the present invention has a wide pH adaptability.

Example 5 measurement of optimum temperature of L-TA Sz-1-2

The enzyme reaction sample was reacted in a shaker at 5-42 ℃ for 2 hours at 200rpm, and de was detected and calculated according to the method for determining de in example 4. Each sample was subjected to not less than 3 replicates and the results averaged.

As shown in FIG. 7, the highest diastereoselectivity was achieved at 37 ℃ of 29.66%, and the yield was higher than that at other temperatures under the same reaction conditions at 37 ℃ so that the temperature was selected as the optimum temperature for the enzymatic reaction under the same conditions. This mild temperature makes the threonine aldolase of the present invention more suitable for industrial applications.

Sequence listing

<110> university of Chongqing

<120> threonine aldolase, gene encoding the same, and use thereof in droxidopa biosynthesis

<130> P1930593-CQD-CQ-XDW

<141> 2019-10-12

<150> 2019104629462

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 343

<212> PRT

<213> Unknown (Unknown)

<400> 1

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 Gln 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> Unknown (Unknown)

<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

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<400> 3

cgcggatcca tgtatagttt taaaaatgat tat 33

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccgctcgagt tatttattag ttaatccact ta 32

Claims

1. An L-threonine aldolase represented by a1 or a 2:

a protein shown as SEQ ID NO. 1;

2. The gene encoding L-threonine aldolase as set forth in claim 1.

3. The encoding gene of claim 2, wherein: the coding gene is any one of the following b1-b 3:

b1. a DNA molecule represented by SEQ ID NO. 2;

b2. a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in b1 and which encodes the L-threonine aldolase of claim 1;

b3. a DNA molecule having more than 90% identity to the DNA molecule defined in b1 or b2 and encoding the L-threonine aldolase of claim 1.

4. A recombinant vector, expression cassette or recombinant cell comprising the coding gene of claim 2 or 3.

5. Use of the L-threonine aldolase of claim 1 for catalyzing a reaction.

6. A catalyst, characterized by: comprising the L-threonine aldolase of claim 1.

7. A method for carrying out a selective aldehyde condensation reaction of a substrate, characterized in that: a substrate is subjected to a catalytic reaction using the L-threonine aldolase of claim 1 or the catalyst of claim 6.

8. The method of claim 7, wherein: the catalytic reaction takes 3, 4-dihydroxy benzaldehyde as a substrate, takes pyridoxal 5-phosphate as a coenzyme and glycine as an auxiliary substrate to synthesize droxidopa.

9. The method of claim 8, wherein: the dosage of the L-threonine aldolase is 100-2000mg/mL, the concentration of the 3, 4-dihydroxybenzaldehyde is 1-100g/L, the concentration of the glycine is 5-100g/L, and the concentration of the 5-pyridoxal phosphate is 0.01-0.2 g/L.

10. The method according to any one of claims 7-9, wherein: the temperature of the catalytic reaction is controlled to be 5-42 ℃, and the stirring speed is 70-300 rpm.