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Amino acid dehydrogenase mutant and application thereof

A technology of amino acid dehydrogenase and leucine dehydrogenase, which is applied in the cross field of chemical catalysis and enzyme catalysis to achieve the effect of improving reaction efficiency

Pending Publication Date: 2021-11-02
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common synthetic methods include: enzymatic synthesis, α,β-dehydroamino acid hydrogenation or cycloaddition, Strecker type reaction, electrophilic or nucleophilic amination and electrophilic or nucleophilic alkylation, etc., but these Synthetic methods all have more or less unfavorable factors in terms of chiral control, reaction conditions and reaction efficiency

Method used

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  • Amino acid dehydrogenase mutant and application thereof
  • Amino acid dehydrogenase mutant and application thereof
  • Amino acid dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Extraction of Bacillus subtilis (B.subtilis) genomic DNA

[0049] Streak and activate B.subtilis frozen at -80°C on LB plate, pick a single colony and inoculate it in 4mL LB liquid medium, cultivate overnight at 37°C, centrifuge at 3000rpm for 5min with a 1.5mL centrifuge tube, discard the supernatant to collect the bacteria , can be repeated several times to obtain a sufficient number of cells; b) extract the Bacillus subtilis genome according to the operating instructions of the TIANGEN Bacteria Genomic DNA Extraction Kit (TIANamp Bacteria DNAKit).

Embodiment 2

[0050] Cloning of embodiment 2 wild-type BsLDH gene

[0051] With the Bacillus subtilis genomic DNA that embodiment 1 obtains as template, carry out PCR reaction, reaction system is as follows:

[0052]

[0053] Amplification program: 94°C, 10min; 34 cycles (94°C: 30s, 45°C: 30s, 72°C: 30s); 72°C, 10min.

[0054] Primer: BsLDH-EcoRI-F CCG GAATTC GATGGAACTTTTTAAATAT

[0055] BsLDH-XhoI-R CCG CTCGAG ACGTCTGCTTAATAC

[0056] Among them, restriction endonuclease cut sites are underlined.

[0057] The DNA fragments amplified by PCR were purified using a gel recovery kit. E.coliDH5α containing the pET-28b-MBP plasmid was cultured overnight in LB liquid medium at 37°C and 200 rpm, and the plasmid was extracted using the reference TIANprep MiniPlasmid Kit.

[0058] The target fragment and plasmid pET-28b-MBP were double-digested with restriction endonucleases EcoRI and XhoI, and the digestion system was as follows:

[0059]

[0060]

[0061] The digested products wer...

Embodiment 3E

[0062] Preparation and transformation of embodiment 3E.coli Rosetta (DE3) competent cells

[0063] a) by CaCl 2 Preparation method Prepare competent cells of E.coli Rosetta (DE3); b) Add 15 μL of T4 ligase reaction solution to 150 μL of competent cell suspension, swirl gently to mix, and ice-bath for 30 minutes; c) Incubate at 42°C Heat shock for 60s, ice bath for 2min, add 800μL LB liquid medium. After static culture at 37° C. for 1 h, shaking culture at 150 rpm for 1 h at the same temperature; d) 150 μL of transformation solution was respectively spread on LB resistance plates.

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Abstract

The invention belongs to the crossing field of chemical catalysis and enzyme catalysis, relates to an amino acid dehydrogenase mutant and application thereof, and in particular relates to organic synthesis, mutation modification of bacillus subtilis leucine dehydrogenase BsLDH, asymmetric reductive ammoniation of a substrate [alpha]-keto acid and a derivative thereof, and application of the amino acid dehydrogenase mutant to preparation of an amino acid dehydrogenase mutant, and a chemical-enzyme method continuous reaction preparation process of the chiral non-natural amino acid. The mutant disclosed by the invention can catalyze stereoselective reductive ammoniation of various [alpha]-keto acid compounds to synthesize non-natural amino acids with various structures. The mutant gene is expressed in E.coli Rosseta (DE3) after being recombined and transformed, and the mutant gene and glucose dehydrogenase (GDH) form a coenzyme circulation system for catalyzing [alpha]-keto acid and derivatives thereof to obtain chiral non-natural amino acid. Meanwhile, a methyl ketone compound is used as a starting raw material, and a chemical enzyme method reaction process for stereoselectively preparing chiral non-natural amino acid is constructed.

Description

technical field [0001] The invention belongs to the cross field of chemical catalysis and enzymatic catalysis, relates to amino acid dehydrogenase mutants and applications thereof, in particular to organic synthesis, mutation transformation of Bacillus subtilis leucine dehydrogenase (leucine dehydrogenase from B.subtilis, BsLDH), bottom Asymmetric reductive amination of α-keto acids and their derivatives, and chemical-enzymatic continuous reaction preparation process of chiral unnatural amino acids. Background technique [0002] Amino acids play an important role in the early days of modern drug discovery, and they are found in antibiotics such as bacitracin and vancomycin and in peptides typified by insulin. Due to the diversity of amino acid compound structures, the increasing acceptance of peptides and modified polypeptide drugs, and the ease of synthesis of amino acids with different side chains, amino acid compounds play an important role in modern medicinal chemistry a...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12P13/04C07C227/08C07C229/36C07C51/16C07C59/21
CPCC12N9/0016C12P13/04C07C227/08C07C51/16C12Y104/01009C07C229/36C07C59/21
Inventor 贾娴欧阳敬平游松韩冰洋刘家岱范多纳张飞霆
Owner SHENYANG PHARMA UNIVERSITY
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