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Leucine dehydrogenase mutant with improved catalytic activity and application of leucine dehydrogenase mutant

A technique for leucine dehydrogenase and mutants, applied in the field of leucine dehydrogenase mutants

Active Publication Date: 2020-10-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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(2014)), L-tert-leucine (Li J, Pan J, Zhang J, et al.Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum[J].Journal of Molecular Catalysis B Enzymatic, 2014, 105(7):11-17), etc.; and there are many literatures to study and optimize the one-pot preparation of leucine dehydrogenase coupling coenzyme cycle and threonine deaminase However, in the process of preparing unnatural amino acids such as L-2-aminobutyric acid, the catalytic activity of leucine dehydrogenase is lower than that of threonine deaminase, resulting in the continuous accumulation of intermediate products and continuous inhibition of leucine dehydrogenation The activity of the enzyme makes leucine dehydrogenase insufficient to catalyze the intermediate product 2-ketobutyrate that is constantly produced, so a leucine dehydrogenase with high catalytic activity is urgently needed

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  • Leucine dehydrogenase mutant with improved catalytic activity and application of leucine dehydrogenase mutant
  • Leucine dehydrogenase mutant with improved catalytic activity and application of leucine dehydrogenase mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Expression of Leucine Dehydrogenase Wild Enzyme

[0038](1) Synthesize and obtain the leucine dehydrogenase whose coding nucleotide sequence is shown in SEQ ID NO.3.

[0039] (2) Construction and transformation of gene expression vector

[0040] The leucine dehydrogenase and the pET-28a vector with the coding nucleotide sequence shown in SEQ ID NO.3 are double-digested with restriction endonucleases BamH I and Hind III, and the products after digestion are 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

[0041] Embodiment 2: the preparation of leucine dehydrogenase mutant

[0042] Specific steps are as follows:

[0043] (1) Preparation of Leucine Dehydrogenase Mutants

[0044] Using the reverse PCR method of the whole plasmid, the oligonucleotide fragments with different numbers of amino acids excised from the C-terminal were designed as upstream and downstream primers for the homology arms, and the recombinant plasmid pET-28a-LeuDH obtained in Example 1 was used as a template to carry out the C-terminal excision transformation, Obtained the mutant carrying the encoding leucine dehydrogenase△ 364-374 , △ 369-374 , △ 373-374 Gene recombinant plasmids pET-28a-LeuDH1~pET-28a-LeuDH3;

[0045] Among them, the mutant △ 364-374 It is obtained by excising the C-terminal residue 364-arginine to 374-glycine with the leucine dehydrogenase shown in SEQ ID NO.1, and the primers used are as follows:

[0046] △ 364-374 -F: 5'-GACAGCAAATGGGTCGCGGATCCATGGTGGAAACCAATGTGGAAG-3' (SEQ ID NO...

Embodiment 3

[0058] Example 3: Expression of Leucine Dehydrogenase Mutants

[0059] 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-LeuDH3 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 with improved catalytic activity and application of the leucine dehydrogenase mutant, and belongs to the technical field of enzyme engineering and microbial engineering. The invention provides a leucine dehydrogenase mutant, which is subjected to C-terminal modification compared with leucine dehydrogenase with an amino acid sequence shown as SEQ ID NO.2, 364-bit arginine to 374-bit glycine of a C-terminal residue are cut off. A leucine dehydrogenase substrate access channel is modified for the first time, the access channel of the substrate is possibly expanded by cutting off the mutant at the C-terminal, so that the substrate is more easily combined with protein, and the leucine dehydrogenase capable of preparing L-2-aminobutyric acidmore efficiently is obtained. The specific enzyme activity of the leucine dehydrogenase mutant [Delta]364-374 provided by the invention is 201U / mg, and is improved by 20% compared with that of wild enzyme.

Description

technical field [0001] The invention relates to a leucine dehydrogenase mutant with improved catalytic activity and application thereof, belonging to the technical fields of enzyme engineering and microbial 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 dehydr...

Claims

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Application Information

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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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