Method for preparing chiral amine through co-immobilization cascade reaction of alcohol dehydrogenase and amine dehydrogenase mediated by silicon dioxide binding peptide

A technology of silica and alcohol dehydrogenase, which is applied in the fields of biochemical equipment and methods, oxidoreductases, and botanical equipment and methods, and can solve the problem that single-enzyme catalysis cannot meet production needs, small specific surface area, and is not conducive to immobilization. The contact between enzyme and substrate molecule, etc.

Pending Publication Date: 2022-04-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing reports on the immobilization of enzymes based on CotB1p are limited to single enzymes and micron-scale silicon-based carriers, and there is almost no research on the use of CotB1p tags to immobilize dual enzymes on the surface of nano-scale silicon-based carriers.
Single-enzyme catalysis cannot meet the production needs of various chemicals in industrial applications
Moreover, the micron-sized immobilized carrier has a small specific surface area, which is not conducive to the contact between the immobilized enzyme and the substrate molecule.

Method used

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  • Method for preparing chiral amine through co-immobilization cascade reaction of alcohol dehydrogenase and amine dehydrogenase mediated by silicon dioxide binding peptide
  • Method for preparing chiral amine through co-immobilization cascade reaction of alcohol dehydrogenase and amine dehydrogenase mediated by silicon dioxide binding peptide
  • Method for preparing chiral amine through co-immobilization cascade reaction of alcohol dehydrogenase and amine dehydrogenase mediated by silicon dioxide binding peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] 1) Pass the gene (SEQ ID NO.1) of CotB1p through (G 4 S) 2 The connecting peptide was fused with the N-terminus of the ADH (SEQ ID NO.2) and AmDH (SEQ ID NO.3) genes respectively to obtain CotB1p-ADH and CotB1p-ADH with gene sequences of SEQ ID NO.4 and SEQ ID NO.5 respectively AmDH, and then subcloning the fused gene into the restriction site between NdeI and XhoI of plasmid pColdII (SEQ ID NO.6) to form recombinant plasmids (pColdII-CotB1p-ADH, pColdII-CotB1p-AmDH) , which was verified by sequencing, transformed into Escherichia coli shuffle T7-B competent cells, and obtained genetically engineered strains;

[0068] 2) The genetically engineered strains are cultured in two stages, first grown to OD at 37°C 600nm= 1.0, then add 0.1M IPTG at 16°C to induce recombinant protein expression for 24 hours, collect the expression strain by centrifugation (4000g, 30min) after induction, and store it at -20°C for later use;

[0069] 3) Resuspend the above obtained strain in l...

Embodiment 2

[0075] 1) Pass the gene (SEQ ID NO.1) of CotB1p through (G 4 S) 2 The connecting peptide was fused with the N-terminus of the ADH (SEQ ID NO.2) and AmDH (SEQ ID NO.3) genes respectively to obtain CotB1p-ADH and CotB1p-ADH with gene sequences of SEQ ID NO.4 and SEQ ID NO.5 respectively AmDH, and then subcloning the fused gene into the restriction site between NdeI and XhoI of plasmid pColdII (SEQ ID NO.6) to form recombinant plasmids (pColdII-CotB1p-ADH, pColdII-CotB1p-AmDH) , which was verified by sequencing, transformed into Escherichia coli shuffle T7-B competent cells, and obtained genetically engineered strains;

[0076] 2) The genetically engineered strains are cultured in two stages, first grown to OD at 37°C 600nm = 1.0, then add 0.1M IPTG at 16°C to induce the expression of CotB1p-ADH for 24 hours, and collect the expression strain by centrifugation (4000g, 30min) after the induction, and store it at -20°C for later use;

[0077] 3) Resuspend the above obtained stra...

Embodiment 3

[0083]1) Pass the gene (SEQ ID NO.1) of CotB1p through (G 4 S) 2 The connecting peptide was fused with the N-terminus of the ADH (SEQ ID NO.2) and AmDH (SEQ ID NO.3) genes respectively to obtain CotB1p-ADH and CotB1p-ADH with gene sequences of SEQ ID NO.4 and SEQ ID NO.5 respectively AmDH, and then subcloning the fused gene into the restriction site between NdeI and XhoI of plasmid pColdII (SEQ ID NO.6) to form recombinant plasmids (pColdII-CotB1p-ADH, pColdII-CotB1p-AmDH) , which was verified by sequencing, transformed into Escherichia coli shuffle T7-B competent cells, and obtained genetically engineered strains;

[0084] 2) The genetically engineered strains are cultured in two stages, first grown to OD at 37°C 600nm = 1.0, then add 0.1M IPTG at 16°C to induce recombinant protein expression for 24 hours, collect the expression strain by centrifugation (4000g, 30min) after induction, and store it at -20°C for later use;

[0085] 3) Resuspend the above obtained strain in l...

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Abstract

The invention relates to a method for preparing chiral amine through co-immobilization cascade reaction of alcohol dehydrogenase and amine dehydrogenase mediated by silicon dioxide binding peptide. Respectively inserting the genes of the silicon dioxide binding peptide into N tail ends of gene sequences of mediated alcohol dehydrogenase and amine dehydrogenase by adopting an overlapping extension PCR (Polymerase Chain Reaction) technology, and constructing a gene engineering strain; purifying and separating the recombinant protein by using a nickel ion metal chelating affinity chromatographic column and a high-concentration imidazole buffer solution; the method comprises the following steps: by taking silicon dioxide nanoparticles as a carrier of an immobilized enzyme, co-anchoring a recombinant protein on the surface of SNPs to obtain an immobilized enzyme CotB1p-ADHamp; cotB1p-AmDH (at) SNPs (single nucleotide polymorphism); and placing the obtained immobilized enzyme in an NH4Cl buffer solution containing NAD < + > and racemic alcohol for reaction to prepare the chiral amine. The prepared double-enzyme co-immobilized catalyst shows relatively high enzyme loading capacity and improved catalytic activity and stability, and has relatively great potential in the aspect of chiral amine production in industrial application.

Description

technical field [0001] The invention relates to a method for preparing chiral amine through co-immobilization cascade reaction of alcohol dehydrogenase (ADH) and amine dehydrogenase (AmDH) mediated by silica binding peptide (CotB1p), belonging to the technical field of biocatalysis. Background technique [0002] Chiral amine refers to a class of compounds containing amino groups in the chiral center, which account for a large proportion of biologically active small molecules, and are also important intermediates for many medicines and pesticides, such as drugs for diabetes (Sitaxel Tin), or pesticides used in agricultural production (glufosinate-ammonium). At present, the synthesis methods of chiral amines mainly include chemical catalytic synthesis, biocatalytic synthesis and chemical-biological combined catalytic synthesis. Among them, chemically catalyzed asymmetric reduction of latent chiral C=N bonds has been widely used in industrial production. However, the chemical...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N15/70C12N15/62C12N15/53C12N15/31C12P41/00C12P13/00
CPCC12N11/14C12N9/0006C12N9/0014C07K14/32C12N15/70C12P41/006C12P13/001C12Y104/99003
Inventor 孙彦刘思董晓燕史清洪
Owner TIANJIN UNIV
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