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Leucine dehydrogenase mutant and application thereof

A technology of leucine dehydrogenase and mutants, which is applied in the field of leucine dehydrogenase mutants and its applications, and can solve problems such as substrate intolerance

Active Publication Date: 2020-09-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And there are many literatures to study and optimize the one-pot preparation of leucine dehydrogenase coupling coenzyme cycle and threonine deaminase. However, in its preparation of unnatural amino acids such as L-2-aminobutyric acid There is the shortcoming of the substrate intolerance of leucine dehydrogenase in the process, therefore, be badly in need of finding a kind of leucine dehydrogenase with strong substrate tolerance in the L-2-aminobutyric acid production process

Method used

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  • Leucine dehydrogenase mutant and application thereof
  • Leucine dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Expression of Leucine Dehydrogenase Wild Enzyme

[0036] (1) Synthesize and obtain the leucine dehydrogenase whose coding nucleotide sequence is shown in SEQ ID NO.1.

[0037] (2) Construction and transformation of gene expression vector

[0038] The leucine dehydrogenase and the pET-28a vector with the coding nucleotide sequence shown in SEQ ID NO.1 are double-digested with restriction endonucleases BamH I and Hind III, and the product after digestion is ligated with Solution I and The ligation product was transformed into Escherichia coli BL21(DE3), and four transformants were picked to extract plasmids BamH I and Hind III for enzyme digestion verification to obtain recombinant Escherichia coli BL21 / pET-28a-LeuDH.

Embodiment 2

[0039] Embodiment 2: the preparation of leucine dehydrogenase mutant

[0040] Specific steps are as follows:

[0041] (1) Preparation of Leucine Dehydrogenase Mutants

[0042] Using the whole plasmid reverse PCR method, the oligonucleotide fragment containing the mutation point was used as the homology arm to design the upstream and downstream primers, and the recombinant plasmid pET-28a-LeuDH obtained in Example 1 was used as the template to carry out site-directed mutation, and the gene carrying the gene encoding LeuDH was obtained. Recombinant plasmids pET-28a-LeuDH1~pET-28a-LeuDH4 of genes of amino acid dehydrogenase mutants K72A, Q361N, R69A, N84L;

[0043] Wherein, the mutation K72A is obtained by mutating the 72nd amino acid of the leucine dehydrogenase shown in SEQ ID NO.2 from lysine to asparagine, and the primers used are as follows:

[0044] K72A-F: 5'-CCGTCTGGCGGCGGGCATGACCTACAAGAATGCCGCGG-3' (SEQ ID NO.7)

[0045] K72A-R: 5'-TCATGCCCGCCGCCAGACGGAGCGCGTCAATCAGCGC-...

Embodiment 3

[0059] Example 3: Expression of Leucine Dehydrogenase Mutants

[0060] The recombinant Escherichia coli BL21 / pET-28a-LeuDH prepared in Example 1 and Example 2 and the recombinant Escherichia coli BL21 / pET-28a-LeuDH1~pET-28a-LeuDH4 were respectively inoculated in LB containing 50 μg / mL kanamycin In the liquid medium, after culturing overnight at 37°C and 180r / min, inoculate 1% of the inoculum into 50mL LB medium, culture at 37°C, rotate at 180r / min, and cultivate until OD600 reaches 0.5-0.8 Then add IPTG with a final concentration of 0.5mM for induction, and the induction temperature is lowered to 25°C. After induction for 8-10h, 4°C, 8000rpm centrifuge for 10min to collect the bacterial cells, take the collected wet bacterial cells, and use 5mL of 50mM pH 7.5 Wash twice with PBS buffer, resuspend in 5mL of 50mM PBS buffer with pH 7.5, oscillate and shake well, and then break under ultrasonic wave, break for 1s, stop for 3s, the total time is 15min. The cell lysate was centrif...

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Abstract

The invention discloses a leucine dehydrogenase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. Compared with leucine dehydrogenasewith the amino acid sequence shown as SEQ ID NO. 2, the leucine dehydrogenase mutant has the advantage that the 72 amino acid is mutated into alanine from lysine. The leucine dehydrogenase mutanthas the advantages that the site-specific mutagenesis is performed on alkaline amino acid residues in a leucine dehydrogenase substrate passage for the first time; the structure and the environment near the possible substrate binding site of the enzyme are modified; and the leucine dehydrogenase for more efficiently preparing L-2-aminobutyric acid is obtained. The leucine dehydrogenase mutant K72Awith the improved substrate tolerance provided by the invention can tolerate 4.5g / L 2-ketobutyric acid; the catalysis activity on the substrate 2-ketobutyric acid is improved by 15 percent.

Description

technical field [0001] The invention relates to a leucine dehydrogenase mutant and its application, belonging to the technical fields of enzyme engineering and microbe engineering. Background technique [0002] L-2-aminobutyric acid has great application value in food, agriculture, biomedicine, etc. For example, it is the direct precursor for the treatment of antiepileptic drugs levopiracetam and buvaracetam, and it is also used for An important chiral precursor for the synthesis of the drug ethambutol hydrochloride for the treatment of tuberculosis. Leucine dehydrogenase (LeuDH, EC 1.4.1.9) belongs to the oxidoreductase family and has been widely used in the preparation of unnatural amino acids, such as L-2-aminobutyric acid (Tao, R., Jiang, Y.,Zhu,F.et al.A one-pot system for production of L-2-aminobutyric acid from L-threonine by L-threonine deaminase and a NADH-regeneration system based on L-leucine dehydrogenase and formate dehydrogenase.Biotechnol Lett 36, 835–841(20...

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
CPCC12N9/0016C12N15/70C12P13/04C12Y104/01009
Inventor 饶志明徐美娟陈佳杰张显杨套伟邵明龙
Owner JIANGNAN UNIV
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