Leucine dehydrogenase mutant, coding gene, vector, engineering bacterium and application thereof

A technology of leucine dehydrogenase and coding gene, which is applied in the field of leucine dehydrogenase mutants, to achieve the effect of improving enzyme activity, good technical support, and simple process

Active Publication Date: 2017-03-15
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There have been reports that leucine dehydrogenase is used to produce L-2-aminobutyric acid, and from the current results, leucine dehydrogenase is also a limiting factor in this production process

Method used

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  • Leucine dehydrogenase mutant, coding gene, vector, engineering bacterium and application thereof
  • Leucine dehydrogenase mutant, coding gene, vector, engineering bacterium and application thereof
  • Leucine dehydrogenase mutant, coding gene, vector, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Gene synthesis of leucine dehydrogenase

[0044] The gene of leucine dehydrogenase is derived from the whole genome sequence of Thermoactinomyces intermedius. In order to express the protein with His-tag after the gene is connected to the vector pET-28b, its stop codon was excised, and its sequence and common restriction endonuclease were compared with the codon preference of B. subtilis 168 as a reference Sequence optimization of the recognition sites BamH I, Xho I, Pst I, Hind III and Nco I, the sequence of the newly designed leucine dehydrogenase gene (leudh) is shown in SEQ ID NO.1, and the sequence of the encoded amino acid is As shown in SEQ ID NO.12, the gene synthesis work was entrusted to Shanghai Xuguan Biotechnology Development Co., Ltd., and the gene synthesis was connected to the cloning vector pET28b.

Embodiment 2

[0045] Example 2: Construction of Leucine Dehydrogenase Recombinant Escherichia coli

[0046] On the basis of Example 1, using PCR technology, using the synthetic nucleotide sequence (shown in SEQ ID NO.1) as a template, leudh-F: 5'-CGTACGCTTCCGAANNNGAGGCGATTGAAG-3' and leudh-R: 5' -CTTCAATCGCCTCNNNTTCGGAAGCGTACG-3' as a primer to amplify the leucine dehydrogenase gene (leudh), and introduce Nco I and Xho I restriction enzyme sites at its 5' end and 3' respectively. The PCR reaction system (50 μL) was: 5 μL of 10×Taqpolymerase buffer, 4 μL of dNTP Mixture; 1 μL of template DNA; 0.5 μL of upstream and downstream primers; 0.5 μL of Taqpolymerase DNA polymerase; 38.5 μL of sterile water. The program of PCR reaction was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 50°C for 45 s, extension at 72°C for 1 min (30 cycles); extension at 72°C for 10 min. Verify with 1% agarose gel electrophoresis and recover the PCR amplified product with 0.8% agarose...

Embodiment 3

[0048] Example 3: Construction of a single point mutant of leucine dehydrogenase

[0049] The recombinant Escherichia coli BL21 / pET-28b-leudh constructed in Example 2 was extracted from the plasmid, and the plasmid pET-28b-leudh was used as a template to design six pairs of mutation primers for site-directed mutation according to the parental sequence.

[0050] L41G-F:5′-CCCTGGGTCCGGC GGT TGGTGGTATGCGTATG-3' and

[0051] L41G-R:5′-CATACGCATACCACC ACC AGCCGGACCCAGGG-3′;

[0052] A61T-F:5′-GATTGAAGATGCTCT ACT CTGGGTCGTGGCATGAC-3′ and

[0053] A61T-R:5′-GTCATGCCACGACCCAG AGT CAGAGCATCTTCAATC-3′;

[0054] L77C-F: 5'-GCTGCAGGTCTGAAC TGT GGTGGCGGCAAAACC-3' and

[0055] L77C-R: 5'-GGTTTTGCCGCCACC ACA GTTCAGACCTGCAGG-3';

[0056] M347G-F: 5’-GAACGTATCGAAATG GGT CGTAAGACCCGCAGAAAGGTG-3' and

[0057] M347G-R: 5'-GTGCTGCGGGTCTTACGNNNCA ACC CGATACGATACGTTC-3';

[0058] Q358T-F: 5’-CACCTTTTCTGCAGGAC ACT CGTAACCTGATCAAC-3' and

[0059] Q358T-R: 5’-GTGCTGCGGGTCTTACG ...

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Abstract

The invention discloses a leucine dehydrogenase mutant, a coding gene, a recombinant vector, a gene engineering bacterium and application thereof. The recombinant Escherichia coli strain with efficiently expressed leucine dehydrogenase has the advantages of high yield, simple technique and the like, is convenient for industrialized application, and can be directly used for producing L-2-aminobutyric acid. After the LeuDH-Q358T mutant fermentation finishes, the total enzyme activity for 2-ketobutyric acid at 35 DEG C is up to 965.7 U / g, and the conversion rate of 2-ketobutyric acid is up to 99%, thereby providing favorable technical supports for large-scale production of L-2-aminobutyric acid.

Description

[0001] (1) Technical field [0002] The invention relates to a leucine dehydrogenase mutant, a leucine dehydrogenase-producing genetically engineered bacterium, a construction method and application thereof, and belongs to the field of genetic engineering. [0003] (2) Technical background [0004] Leucine dehydrogenase (Leu DH for short, EC 1.4.1.9), which can catalyze the conversion of α-butyronic acid into L-2-aminobutyric acid, is one of the important biocatalysts in the field of pharmaceutical production, especially It plays a key role in the process of preparing L-2-aminobutyric acid through biotransformation. [0005] L-2-aminobutyric acid (L-ABA) is a non-natural chiral α-amino acid, which is an important chemical raw material and pharmaceutical intermediate. For example, it is used in the production of the anti-epileptic drug levetiracetam and the anti-tuberculosis drug ethambutol hydrochloride. [0006] There have been reports that leucine dehydrogenase is used to p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/0016C12P13/04C12Y104/01009
Inventor 徐建妙郑裕国柳志强傅芳田胡海峰
Owner ZHEJIANG UNIV OF TECH
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