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Threonine deaminase mutant as well as preparation method and application thereof

A threonine deaminase and mutant technology, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, and applications, can solve problems such as poor stability, increase costs, limit the service life of biocatalysts, etc. Industrialization, the effect of improving thermal stability

Active Publication Date: 2019-01-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there have been reports of threonine deaminase being applied to the synthesis of L-2-aminobutyric acid, but its stability is poor, which limits the service life of the biocatalyst, limits the catalytic efficiency, and increases the cost in industrial production. cost

Method used

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  • Threonine deaminase mutant as well as preparation method and application thereof
  • Threonine deaminase mutant as well as preparation method and application thereof
  • Threonine deaminase mutant as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: Construction of threonine deaminase recombinant bacteria

[0021] Threonine deaminase derived from Escherichia coli K12 was selected, using PCR technology, using the E.coli K12 genome as a template, ilvA-F: 5'-GGAATTCCATATGGCTGACTCGCAACCCCT G-3' and ilvA-R: 5'-CCGCTCGAGTTAACCCGCCAAAAAGAAC-3 ' as a primer to amplify threonine deaminase, and introduce Nde I and Xho I restriction enzyme sites at its 5' end and 3' end, respectively. The PCR reaction system (50 μL) was: 10 μL of 5×PrimeSTAR GXL buffer, 1 μL of PrimeSTAR GXL DNA Polymerase, 4 μL of 2.5mM dNTPs, 1 μL of template DNA, 1 μL of upstream and downstream primers, and 32 μL of sterile water. The PCR reaction conditions were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 68°C for 2 min, and 30 cycles; extension at 68°C for 10 min. Verify and recover the PCR amplification product with 1% agarose gel electrophoresis, the result is amplified to t...

Embodiment 2

[0023] Embodiment 2: Directed evolution mutation of threonine deaminase gene

[0024] Extract the recombinant Escherichia coli BL21(DE3) / pET28a(+)-ilvA plasmid in Example 1, use the plasmid pET28a(+)-ilvA as a template, and use Primers-F: 5'-CGCGGATCCATGGCTGACTCG A-3', Primers- R: 5'-CCCAAGCTTAACCCGCCAAAAAGAAC-3' is used as a primer to introduce replication errors into the polymerase chain reaction by adding divalent manganese ions and divalent magnesium ions. The PCR reaction system for directed evolution is: 5mM MnCl 2 1 μL, Taq polymerase 0.5 μL, 10× buffer 10 μL, 25mM MgCl 2 14 μL, 4 μL of dNTP Mixture, 1 μL of upper and lower primers, 0.5 μL of template DNA (50ng / μL) and 18 μL of sterile water. PCR reaction conditions for directed evolution: 94°C for 5min; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 3min; 72°C for 10min.

[0025] The PCR products were purified and treated with restriction endonucleases BamH I and Hind III at 37°C for 3 hours. The PCR product a...

Embodiment 3

[0028] Example 3: Escherichia coli K12 Threonine Deaminase Gene Multiple Sequence Alignment and Computer Simulation Mutation

[0029] Through BLAST search, it was found that threonine deaminase belongs to Trp-synth-beta II superfamily (accession ID: cd01562). And found four different strains of thermostable threonine deaminase. They are Deinococcus-Thermus (accession ID: WP_058978819.1), Chloroflexus sp.MS-G (accession ID: WP_031459137.1), Hydrogenophilales bacterium (accession ID: OGU19679.1) and Pseudomonas aeruginosa (accession ID: NP_1500 ). According to the results of amino acid sequence alignment (such as image 3 ), and found 9 possible amino acid residues, namely: Val131, Ala114, Phe157, Ala193, Glu240, Val281, Gly323, Ser443, Phe510. The "weighted mutation energy" of the mutant strains Val131Phe, Ala114Pro, Phe157Tyr, Ala193Ile, Glu240Asp, Val281Thr, Gly323Arg, Ser443Arg, and Phe510Leu was calculated by computer simulation mutation, and the mutation type with the s...

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Abstract

The invention discloses a threonine deaminase mutant as well as a preparation method and application thereof. The mutant is prepared by carrying out single-point mutation or multi-point mutation on amino acids on a site 14, a site 323, a site 344, a site 449 and a site 510 in an amino acid sequence represented by SEQ ID NO.1. Compared with wild threonine deaminase, threonine deaminase provided bythe invention has the advantages that the stability is extremely high, and the service life of threonine deaminase in the industrial production can be greatly prolonged. A G323D / F510L / T344A mutant isapplied to the production of L-2-aminobutyric acid, the conversion rate of L-threonine can reach 99%, and the addition amount of NAD<+> can be decreased to 0.04g / L, so that a good technical support isprovided for the large-scale production of L-2-aminobutyric acid.

Description

technical field [0001] The invention relates to a threonine deaminase mutant, a threonine deaminase-producing genetically engineered bacterium, a construction method and application thereof, and belongs to the field of genetic engineering. Background technique [0002] Threonine deaminase (TD for short, EC 4.3.1.19), which catalyzes the deamination of L-threonine to generate α-ketobutyrate, is a key step in the metabolic pathway of isoleucine. α-Ketobutyric acid is an important raw material for the production of L-2-aminobutyric acid, isoleucine, D-2-hydroxybutyric acid and n-propanol, especially L-2-aminobutyric acid, which is used as a hand in the treatment of epilepsy Sexual drugs levetiracetam and brivaracetam, anti-tuberculosis drug ethambutol hydrochloride, and endothelin anti-factor, an important precursor of azoxythricin antibiotics, have received extensive attention. [0003] At present, there have been reports that threonine deaminase has been applied to the synth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12P13/04C12Y403/01019
Inventor 林建平王莹朱力乔沛薛海龙李国四吴绵斌杨立荣
Owner ZHEJIANG UNIV
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