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Threonine aldolase, coding gene thereof, and application of threonine aldolase to biosynthesis of L-threo-DOPS

A technology of threonine aldolase and coding gene, applied to L-threonine aldolase, its coding gene and application field in droxidopa biosynthesis, can solve the problem of threonine aldolase stability Poor chirality, insufficient chiral selectivity, limited application, etc.

Active Publication Date: 2019-12-20
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the poor stability of threonine aldolase, the chiral selectivity, especially the insufficient chiral selectivity of β-hydroxyl, limits the application of this enzyme in chiral synthesis.

Method used

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  • Threonine aldolase, coding gene thereof, and application of threonine aldolase to biosynthesis of L-threo-DOPS
  • Threonine aldolase, coding gene thereof, and application of threonine aldolase to biosynthesis of L-threo-DOPS
  • Threonine aldolase, coding gene thereof, and application of threonine aldolase to biosynthesis of L-threo-DOPS

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the discovery of L-TA Sz-1-2 gene

[0053] Fecal samples from healthy black bears in the Sichuan Black Bear Conservation and Incubation Base were collected using sterilized medicine spoons, stored on dry ice and transported back to the laboratory. The total DNA of black bear feces was extracted using the Qiagen Fecal DNA Genome Extraction Kit, and submitted to Shanghai Meiji Biomedical Technology Co., Ltd. for metagenomic sequencing. Compared with the existing Escherichia coli L-threonine aldolase (E.coli.L-TA) coding gene sequence (Genbank accession number: AOM47009.1) and metagenomic sequencing data, a new The L-threonine aldolase gene is named as L-TA Sz-1-2, and its nucleotide sequence is shown in SEQ ID NO: 2. The open reading frame from the 1st position to the 1032nd position at the 5' end of sequence 2 is an open reading frame, and the open reading frame part is 1032bp, and the amino acids of the L-TA Sz-1-2 protein encoded by it are shown in seque...

Embodiment 2

[0054] Cloning of embodiment 2, L-TA Sz-1-2 gene

[0055] 1) Primer design: design primers for the L-TA Sz-1-2 gene sequence obtained by sequencing, and the nucleotide sequences of the primers are as follows:

[0056] Sz-1-2-BamHI-F: 5′-CGC GGATCC ATGTATAGTTTTAAAAATGATTATA-3' (Sequence 3 in the Sequence Listing),

[0057] Sz-1-2-XhoI-R: 5′-CCG CTCGAG TTATTGTACTTCATCTAATAGCAT-3' (SEQ ID NO: 4 in the Sequence Listing).

[0058] 2) PCR amplification: the total DNA of black bear feces was used as a template, and the primers obtained in step 1) were used to amplify according to the following PCR system and procedure.

[0059] PCR system: template DNA is 0.5 μL, Sz-1-2-BamHI-F (10 μM) is 2 μL, Sz-1-2-XhoI-R (10 μM) is 2 μL, 0.1% BSA is 3 μL, Prime STAR HS DNA Polymerase It is 25μL, add ddH 2 O to PCR system is 50 μL.

[0060] The PCR program is as follows:

[0061] a. Pre-denaturation at 94°C for 5 minutes;

[0062] b. Denaturation at 98°C for 10 sec, annealing at 48°C for...

Embodiment 3

[0069] Embodiment 3, expression, extraction and purification of L-TA Sz-1-2 gene

[0070] 1. Construction of the connection vector: the full-length sequence of the L-TA Sz-1-2 gene was connected to the vector pGEX-6p-2.

[0071] The ligation system includes: 6 μL for double-digested L-TA Sz-1-2 gene, 1 μL for T4 DNA Ligase, 2 μL for vector pGEX-6p-2, 1 μL for 10×T4 Ligase buffer, 10 μL for the system, and 16 °C for 12 hours.

[0072] 2. To transform E.coli DH5α competent cells, the steps are as follows:

[0073] 1) Competent cells E.coli DH5α were placed on ice to thaw.

[0074] 2) Add the connection system obtained in step 1 into the melted E.coli DH5α competent, and keep it on ice for 30 minutes.

[0075] 3) Heat treatment at 42°C for 90s.

[0076] 4) Stand on ice for 2 minutes.

[0077] 5) Add 600 μL of anti-antibody-free LB medium, shaker temperature 37° C., shaker speed 150 rpm, time 45 min.

[0078] 6) Aspirate 200 μL of bacterial solution and apply it on ampicillin...

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Abstract

The invention relates to threonine aldolase, a coding gene thereof, and an application of the threonine aldolase to biosynthesis of L-threo-DOPS, and belongs to the field of a biological technology. The threonine aldolase is protein as shown in SEQID NO.1, or protein having the same functions, obtained through replacing and / or deleting and / or adding one or more amino acid residues to an amino acidsequence as shown in SEQID NO.1. The threonine aldolase can use benzaldehyde with replaced 3- / 4- hydroxyl as a substrate, 5-pyridoxal phosphate as a coenzyme and glycine as an assistance substrate. Under the appropriate condition and in an appropriate medium, an enzyme catalysis reaction is performed, and biosynthesis is performed to obtain the L-threo-DOPS (L-threo-DOPS). The non-antipode selectivity of the method can achieve 30%. The threonine aldolase having high selectivity has important significance for biocatalysis of the L-threo-DOPS.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to L-threonine aldolase, its coding gene and its application in droxidopa biosynthesis. Background technique [0002] Droxidopa (L-threo-DOPS) is a new anti-Parkinson's disease drug. For improving gait stiffness and orthostatic dizziness caused by Parkinson's disease; improving orthostatic hypotension, orthostatic dizziness and fainting caused by Shy-Drager syndrome or familial amyloid neuropathy; improving hemodialysis patients due to orthostatic Dizziness and fatigue caused by hypotension. [0003] At present, the synthesis methods of droxidopa mainly include chemical synthesis and enzyme catalysis. Among them, 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 highly toxic substances are used in the react...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12P13/04
CPCC12N9/88C12P13/04C12Y401/02005
Inventor 赵文艳杨碧玲潘银平倪德春王伯初戚娜祝连彩
Owner CHONGQING UNIV
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