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Leucine dehydrogenase mutant and construction method and application thereof

A technology of leucine dehydrogenase and construction method, which is applied in the field of leucine dehydrogenase mutants and its construction, and can solve problems such as inability to catalyze chiral amino acids

Active Publication Date: 2020-10-30
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although leucine dehydrogenase has a wide range of applications, due to the limitation of its own substrate spectrum, leucine dehydrogenase can only catalyze the synthesis of a few chiral amino acids such as L-leucine, and cannot catalyze Synthesis of other chiral amino acids

Method used

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  • Leucine dehydrogenase mutant and construction method and application thereof
  • Leucine dehydrogenase mutant and construction method and application thereof
  • Leucine dehydrogenase mutant and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Preparation of Leucine Dehydrogenase Mutants

[0050] The leucine dehydrogenase derived from Bacillus pumilus was used as the wild type, that is, WT EsiLeuDH.

[0051] (1) Ligate WT EsiLeuDH DNA with plasmid pET28a to obtain a recombinant vector.

[0052] (2) Obtain mutant K89T:

[0053] Using the DNA of the recombinant vector as a template, K89 saturated-IR (the nucleotide sequence is shown in SEQ ID NO.2, specifically: GATGATGACCGCNNNCCCG) and K89 saturated-IF (the sequence is shown in SEQ ID NO.3, specifically: : GCGGGNNNGCGGTCATCAT) was used as a primer to carry out PCR site-directed saturation mutation to obtain a PCR product. Among them, the PCR reaction system is as follows: 1 μL of WT EsiLeuDH DNA at 15 ng / μL, 1 μL of mutant primers IF and IR (10 μM), 15 μL of 2X Trans startfastPfu, and ddH2O supplementation system to 30 μL. PCR reaction program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 20 s, annealing at 60°C for 20 s, extension at 72°C f...

Embodiment 2

[0061] Expression and purification of mutants

[0062] The PCR product obtained in Example 1 was digested with DPN1 enzyme at 37° C. for 2 h. After the digestion treatment, the product was directly transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method to obtain recombinant strains. Spread the obtained recombinant strains on a plate containing Kana antibiotics, and place the plate in a 37°C incubator for 12 hours. After the colonies grow, pick the colonies for PCR colony verification and mutation library screening, followed by liquid culture. A portion of the bacterial culture was sent for sequencing. PCR colony verification and sequencing results showed that mutant A122G and K89T genes had been constructed and successfully introduced into Escherichia coli BL21(DE3).

[0063] The obtained recombinant strain was expanded and cultivated, and the expression of the mutant protein was induced by the chemical reagent IPTG induction method. After the indu...

Embodiment 3

[0066] Enzyme activity assay of leucine dehydrogenase

[0067] The WT purified protein, K89T purified protein and A122G purified protein obtained in Example 2 were subjected to substrate spectrum determination. The substrate spectrum determination system: measured by a microplate reader, the total volume of the reaction system was 220 μL, and the optical path was 0.5 cm. After proper dilution of amino acid dehydrogenase, take 10 μL, 4mM reaction substrate (any one of the 20 kinds of amino acids in the laboratory) 50 μL, 20mM coenzyme NAD+11 μL, 0.2M, pH9.5 glycine-sodium hydroxide 149 μL of buffer solution; measure the increase in NADH concentration at a wavelength of 340 nm, and the enzyme activity of one unit of leucine dehydrogenase is defined as the amount of enzyme required to reduce 1 μmol NAD+ within 1 min.

[0068] Enzyme activity assay principle:

[0069] Enzyme activity (U / mL) = ΔA / Δt×Vt / (Vs×L×ε);

[0070] Specific enzyme activity (U / mg) = enzyme activity (U / mL) / ...

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Abstract

The invention provides a leucine dehydrogenase mutant and a construction method and application thereof, and belongs to the technical field of gene engineering. The mutant disclosed by the invention is obtained by mutating an amino acid sequence of wild leucine dehydrogenase; the 89th lysine of the amino acid sequence of the wild type leucine dehydrogenase is subjected to saturated mutation to form NNN or the 122th alanine is subjected to site-specific mutagenesis to form glycine, and the amino acid sequence of the wild type leucine dehydrogenase is shown as SEQ ID NO. 1. Experimental resultsshow that the K89T leucine dehydrogenase has certain activity on Asn and Thr, the A122G leucine dehydrogenase has certain activity on Phe, His and Met, and the wild leucine dehydrogenase has no activity on the amino acids completely; in addition, the enzyme activity of the K89T leucine dehydrogenase on Asp oxidative deamination reaction is twice that of wild leucine dehydrogenase.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a leucine dehydrogenase mutant and its construction method and application. Background technique [0002] Leucine dehydrogenase (LeuDH) belongs to the short-chain dehydrogenase family, which catalyzes the reductive amination of the natural substrate 4-methyl-2-oxopentanoic acid to obtain L-leucine, which can reversibly catalyze L Oxidative deamination of --type amino acids to synthesize α-keto acids. At present, leucine dehydrogenase has been widely used in the industrial production of chiral amino acids. Leucine dehydrogenase exists widely in natural organisms. The NCBI database retrieves its sources. There are 110,000 sources in total, 1,757 animal sources, 108,864 bacterial sources, 2,221 fungal sources, and a total of 5,000 other sources. At present, leucine dehydrogenase has been widely used in the catalytic synthesis of chiral amino acids. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/70C12R1/19
CPCC12N9/0016C12Y104/01009C12N15/70
Inventor 敬科举熊伟申玉姣卢英华
Owner XIAMEN UNIV
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